A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

Science.gov (United States)

First, Eric A

2015-08-15

A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

2016-01-01

for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

Science.gov (United States)

Sehgal, Pankaj; Olesen, Claus; Møller, Jesper V

2016-01-01

Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans

NARCIS (Netherlands)

van Doorn, H. Rogier; Koelewijn, Rob; Hofwegen, Henk; Gilis, Henk; Wetsteyn, Jose C. F. M.; Wismans, Pieter J.; Sarfati, Claudine; Vervoort, Tony; van Gool, Tom

2007-01-01

A homemade enzyme-linked immunosorbent assay (ELISA) (Academic Medical Center ELISA [AMC-ELISA]) and a dipstick assay for the detection of anti-Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA [IVD Research, Inc.

Assay strategies and methods for phospholipases

International Nuclear Information System (INIS)

Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

1991-01-01

Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

Science.gov (United States)

Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

2017-06-28

The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti- Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

International Nuclear Information System (INIS)

Yin, Si-Tao; Huang, Hao; Zhang, Yu-Hang; Zhou, Zi-Ren; Song, Ai-Xin; Hong, Fa-Shui; Hu, Hong-Yu

2011-01-01

Highlights: ► A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. ► We have established an activity assay for ubiquitin C-terminal hydrolases. ► This assay can be applicable to other deubiquitinating enzymes. -- Abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub F45W -Xaa) and di-ubiquitin chains (Ub F45W -diUb). After removal of the intact substrate by Ni 2+ -NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub F45W product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

DEFF Research Database (Denmark)

Sørensen, A.H.; Ahring, B.K.

1997-01-01

An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...... in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test, The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high...

Lateral flow assays

NARCIS (Netherlands)

Posthuma-Trumpie, G.A.; Amerongen, van A.

2012-01-01

A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

Science.gov (United States)

Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

2014-07-01

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Battery operated preconcentration-assisted lateral flow assay.

Science.gov (United States)

Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

2017-07-11

Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

Directory of Open Access Journals (Sweden)

Vermeulen Michiel

2004-01-01

Full Text Available Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

Comparison of the direct enzyme assay method with the membrane ...

African Journals Online (AJOL)

Comparison of the direct enzyme assay method with the membrane filtration technique in the quantification and monitoring of microbial indicator organisms – seasonal variations in the activities of coliforms and E. coli, temperature and pH.

High sensitivity, high surface area Enzyme-linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Singh, Harpal; Morita, Takahiro; Suzuki, Yuma; Shimojima, Masayuki; Le Van, An; Sugamata, Masami; Yang, Ming

2015-01-01

Enzyme-linked immunosorbent assays (ELISA) are considered the gold standard in the demonstration of various immunological reactions with an application in the detection of infectious diseases such as during outbreaks or in patient care. This study aimed to produce an ELISA-based diagnostic with an increased sensitivity of detection compared to the standard 96-well method in the immunologic diagnosis of infectious diseases. A '3DStack' was developed using readily available, low cost fabrication technologies namely nanoimprinting and press stamping with an increased surface area of 4 to 6 times more compared to 96-well plates. This was achieved by stacking multiple nanoimprinted polymer sheets. The flow of analytes between the sheets was enhanced by rotating the 3DStack and confirmed by Finite-Element (FE) simulation. An Immunoglobulin G (IgG) ELISA for the detection of antibodies in human serum raised against Rubella virus was performed for validation. An improved sensitivity of up to 1.9 folds higher was observed using the 3DStack compared to the standard method. The increased surface area of the 3DStack developed using nanoimprinting and press stamping technologies, and the flow pattern between sheets generated by rotating the 3DStack were potential contributors to a more sensitive ELISA-based diagnostic device.

Photosynthetic carboxylating enzymes in Phaeodactylum tricornutum: assay methods and properties

Energy Technology Data Exchange (ETDEWEB)

Mukerji, D [Bigelow Lab. for Ocean Sciences, West Boothbay Harbor, ME; Morris, I

1976-01-01

Rapid freezing (in liquid nitrogen) of the marine diatom Phaeodactylum tricornutum Bohlin followed by thawing permits a convenient and sensitive measurement of the activities of carboxylating enzymes without the need to prepare a cell-free extract. Using this method, the properties of RuDP and PEP carboxylases have been compared with those assayed in cell-free extracts. The most significant difference was in the Michaelis' constants (K/sub m/'s), the values being lower in the freeze/thaw assay. The absolute rate of carbon-dioxide fixation by the enzymes was less than the rate of photosynthesis by the intact alga. Significantly, the activity of PEP carboxylase was comparable (in some experiments, greater) to that of RuDP carboxylase. The significance of this and the possibility of an enzymatic approach to measurements of marine primary productivity are discussed.

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

Science.gov (United States)

Florea, Mara; Nau, Werner M

2010-03-07

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Konstantinou, George N

2017-01-01

Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.

Science.gov (United States)

McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D

2013-01-01

A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Bergmann, S M; Wang, Q; Zeng, W; Li, Y; Wang, Y; Matras, M; Reichert, M; Fichtner, D; Lenk, M; Morin, T; Olesen, N J; Skall, H F; Lee, P-Y; Zheng, S; Monaghan, S; Reiche, S; Fuchs, W; Kotler, M; Way, K; Bräuer, G; Böttcher, K; Kappe, A; Kielpinska, J

2017-11-01

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs. © 2017 John Wiley & Sons Ltd.

Bluetongue virus: comparative evaluation of enzyme-linked immunosorbent assay, immunodiffusion, and serum neutralization for detection of viral antibodies.

OpenAIRE

Poli, G; Stott, J; Liu, Y S; Manning, J S

1982-01-01

Comparative studies on the detection of bovine serum immunoglobulin G antibodies to bluetongue virus with an enzyme-linked immunosorbent assay, an immunodiffusion method, and a serum neutralization assay demonstrated complete concordance between the enzyme-linked immunosorbent assay and the serum neutralization assay results. However, the immunodiffusion method failed to detect bluetongue virus antibody in a substantial number of sera found to possess bluetongue virus immunoglobulin G with th...

Development of a solid-phase assay for measurement of proteolytic enzyme activity

International Nuclear Information System (INIS)

Varani, J.; Johnson, K.; Kaplan, J.

1980-01-01

A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media

International Nuclear Information System (INIS)

Rucklidge, G.J.; Milne, G.

1990-01-01

A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

Directory of Open Access Journals (Sweden)

A. M. Marassá

2008-01-01

Full Text Available The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Enzyme-linked immunosorbent assays for Z-DNA.

OpenAIRE

Thomas, M J; Strobl, J S

1988-01-01

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e....

Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin

DEFF Research Database (Denmark)

Petersen, H. H.; Nielsen, J. P.; Jensen, A.L.

2001-01-01

An enzyme-linked immunosorbent assay for quantification of haptoglobin in porcine serum was evaluated. Tbe detection limit when expressed as the estimated concentration of a blank sample was 0.0003 mg/ml. The precision of the assay was acceptable with intra-assay coefficients of variation below 4...... % and inter-assay coefficient of variation below 5 % for serum concentrations ranging from 1.0 mg/ml and above. For samples with a concentration below 0.8 mg/ml, the inter-assay coefficient of variation was above 10 %. The assay maintained linearity under dilution. Recovery was proportional. Haemolysis.......2. The maximum allowable analytical imprecision was 2.6 % and the maximum analytical inaccuracy was 9.9 %. The number of samples required to determine, the true haptoglobin value in an individual pig when accounting for the day-to-day fluctuation was 5. In conclusion, the haptoglobin assay was found...

Sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

International Nuclear Information System (INIS)

Stack, G.; Fricker, L.D.; Snyder, S.H.

1984-01-01

A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of 3 H-labeled substrate and product in chloroform. The substrates 3 H-benzoyl-Phe-Ala-Arg or 3 H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, 3 H-benzoyl-Phe-Ala or 3 H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate

Sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

Energy Technology Data Exchange (ETDEWEB)

Stack, G.; Fricker, L.D.; Snyder, S.H.

1984-01-09

A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of /sup 3/H-labeled substrate and product in chloroform. The substrates /sup 3/H-benzoyl-Phe-Ala-Arg or /sup 3/H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, /sup 3/H-benzoyl-Phe-Ala or /sup 3/H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

Science.gov (United States)

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II

Directory of Open Access Journals (Sweden)

Kanchanmala Deshpande

2010-06-01

Full Text Available A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx and horseradish peroxidase (HRP for determination of mercury Hg(II in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II was found to be linear in the range 5–500 pg.mL−1 with 3% CVin inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II was measurable as low as 1 pg.mL−1 within15 min. About 10-fold more specificity of the developed assay for Hg(II analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II and lead Pb(II. Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II, Cd(II and Pb(II, inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%.

THE SPOROZOITE ENZYME-LINKED IMMUNOSORBENT ASSAY : APPLICATION IN MALARIA EPIDEMIOLOGY

Directory of Open Access Journals (Sweden)

Michael J. Bangs

2012-09-01

Full Text Available Recent biotechnological breakthroughs have led to the development of various methods for detection and identification of human pathogens in their vectors. Monoclonal antibodies produced against malaria sporozoite antigens have permitted the development of several sensitive, species specific immunological tests (IFA, IRMA, ELIS A. One of these, a two-site enzyme-linked immunosorbent assay (ELIS A has been developed as a useful epidemiological tool in the identification of malaria-infected mosquitoes. This method employs highly species specific monoclonal antibodies that recognize the repetitive immunodominant epitope of the circumsporozoite (CS protein. Monoclonal antibodies have been developed for all four species of human malaria The key feature of the ELISA technique is the use of an enzyme indicator for an immunological reaction. The antigen capture or "sandwich" ELISA configuration uses the purified monoclonal both as the solid phase and, conjugated to enzyme, as a marker for the presence of CS protein in a mosquito homogenate incubated in the wells of a microtitration plate. This technology has shown advantages over other methods for epidemiological data collection. Mosquitoes can be caught, dried and stored until a time convenient for examination. The sporozoite rate by Plasmodium species can be identified easily, and when combined with the man-biting rate provides the sporozoite inoculation rate, an important entomologic estimate of the number of potential infective bites a person could expect over a given period of time. Presently, mosquitoes can be tested individually or pooled up to 20 anophe lines. The assay is sensitive enough to detect 1 infected mosquito per pool or as few as 25 sporozoites per 50 pi of mosquito extract. Basic principles and procedures are covered concerning solid substrate, adsorption to solid substrate, buffers and wash solutions, conjugates and enzyme substrates. The advantages and limitations of this technique

Development of an enzyme-linked immunosorbent assay method to detect mustard protein in mustard seed oil

NARCIS (Netherlands)

Koppelman, S.J.; Vlooswijk, R.; Bottger, G.; Duijn, G. van; Schaft, P. van der; Dekker, J.; Bemgen, H. van

2007-01-01

An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the

Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

Science.gov (United States)

Groome, N P

1981-10-01

The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism

Czech Academy of Sciences Publication Activity Database

Pavelka, Stanislav

2014-01-01

Roč. 63, Suppl.1 (2014), S133-S140 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) 7AMB12SK158; GA ČR(CZ) GA304/08/0256 Institutional support: RVO:67985823 Keywords : enzyme * metabolism * radiometric assay * thyroid hormone Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.293, year: 2014

Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

Science.gov (United States)

Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

2012-08-21

Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

Science.gov (United States)

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (assay variability assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

High frequency lateral flow affinity assay using superparamagnetic nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Lago-Cachón, D., E-mail: dlagocachon@gmail.com [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Rivas, M., E-mail: rivas@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Martínez-García, J.C., E-mail: jcmg@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Oliveira-Rodríguez, M., E-mail: oliveiramyriam@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); Blanco-López, M.C., E-mail: cblanco@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); García, J.A., E-mail: joseagd@uniovi.es [Dpto. de Física, Universidad de Oviedo, Escuela de Marina, Campus de Viesques, 33204 Gijón (Spain)

2017-02-01

Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies. - Highlights: • A method for quantification of Lateral Flow Assays is proposed. • MNP induce an increase of the impedance on a RF-current carrying copper sensor. • Magnetic nanoparticles (MNP) can be detected flowing over the sensing element.

High frequency lateral flow affinity assay using superparamagnetic nanoparticles

International Nuclear Information System (INIS)

Lago-Cachón, D.; Rivas, M.; Martínez-García, J.C.; Oliveira-Rodríguez, M.; Blanco-López, M.C.; García, J.A.

2017-01-01

Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies. - Highlights: • A method for quantification of Lateral Flow Assays is proposed. • MNP induce an increase of the impedance on a RF-current carrying copper sensor. • Magnetic nanoparticles (MNP) can be detected flowing over the sensing element.

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

Directory of Open Access Journals (Sweden)

Hui Jian

2016-07-01

Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

NARCIS (Netherlands)

Groen, J; Van Dijk, G; Niesters, H G; Van Der Meijden, W I; Osterhaus, A D

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

Science.gov (United States)

Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

2018-01-01

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

Science.gov (United States)

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

International Nuclear Information System (INIS)

Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

1988-01-01

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis

Assaying Oxidative Coupling Activity of CYP450 Enzymes.

Science.gov (United States)

Agarwal, Vinayak

2018-01-01

Cytochrome P450 (CYP450) enzymes are ubiquitous catalysts in natural product biosynthetic schemes where they catalyze numerous different transformations using radical intermediates. In this protocol, we describe procedures to assay the activity of a marine bacterial CYP450 enzyme Bmp7 which catalyzes the oxidative radical coupling of polyhalogenated aromatic substrates. The broad substrate tolerance of Bmp7, together with rearrangements of the aryl radical intermediates leads to a large number of products to be generated by the enzymatic action of Bmp7. The complexity of the product pool generated by Bmp7 thus presents an analytical challenge for structural elucidation. To address this challenge, we describe mass spectrometry-based procedures to provide structural insights into aryl crosslinked products generated by Bmp7, which can complement subsequent spectroscopic experiments. Using the procedures described here, for the first time, we show that Bmp7 can efficiently accept polychlorinated aryl substrates, in addition to the physiological polybrominated substrates for the biosynthesis of polyhalogenated marine natural products. © 2018 Elsevier Inc. All rights reserved.

Continuous-flow chemiluminometric determination of some tetracyclines

International Nuclear Information System (INIS)

Syropoulos, A.B.; Calokerinos, A.C.

1991-01-01

Chemiluminescence is found to be generated by action of lucigenin or hexacyanoferrate(III) on tetracyclines. The reaction with lucigenin exhibits chemiluminescence after alkaline degradation of tetracyclines to the corresponding iso derivatives. The reaction with hexacyanoferrate (III) occurs after acidic degradation of tetracyclines to corresponding anhydro derivatives. The chemiluminescence reaction takes place in alkaline medium, and allows the development of a continuous-flow method for the determination of 1.00-10.0 μgml -1 oxytetracycline and doxycycline. When applied to commercial formulations, the procedure was relatively free from interferences from common excipients. The results obtained for the assay of dosage forms compared well with those obtained by the official methods and demonstrated good accuracy and precision. (author). 32 refs.; 5 figs.; 6 tabs

Continuous-flow chemiluminometric determination of some tetracyclines

Energy Technology Data Exchange (ETDEWEB)

Syropoulos, A B; Calokerinos, A C [University of Athens (greece). Laboratory of Analytical Chemistry

1991-12-24

Chemiluminescence is found to be generated by action of lucigenin or hexacyanoferrate(III) on tetracyclines. The reaction with lucigenin exhibits chemiluminescence after alkaline degradation of tetracyclines to the corresponding iso derivatives. The reaction with hexacyanoferrate (III) occurs after acidic degradation of tetracyclines to corresponding anhydro derivatives. The chemiluminescence reaction takes place in alkaline medium, and allows the development of a continuous-flow method for the determination of 1.00-10.0 {mu}gml{sup -1} oxytetracycline and doxycycline. When applied to commercial formulations, the procedure was relatively free from interferences from common excipients. The results obtained for the assay of dosage forms compared well with those obtained by the official methods and demonstrated good accuracy and precision. (author). 32 refs.; 5 figs.; 6 tabs.

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

Directory of Open Access Journals (Sweden)

Supaporn Kradtap Hartwell

2012-01-01

Full Text Available Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

Enzyme-immuno assay for total estrogens and human placental lactogen. Comparison with radio-immuno-assay in normal pregnancy-monitoring

International Nuclear Information System (INIS)

Raichvarg, D.; Tallet, F.; Lajeunie, E.; Bonnaire, Y.; Danglas, P.

1980-01-01

The concentrations of estrogens (E) and human placental lactogen (HLP) are estimated in sera by radio immuno-assay (RIA) and enzyme-immuno-assay (EIA). Statistical data indicate mean intra-assay variation coefficients of 7% and 12% for E and HLP tests, respectively. The correlation coefficient (RIA/EIA) are found higher than 0,9% for both hormonal assays. The dilution curves obtained by RIA and EIA are similar. However, Student'test gives a significant difference for E determination. In fact, total E and E 3 only are measured by EIA and RIA, respectively. In most cases biological interferences are negligible except for HLP in presence of higher protein or haemoglobin levels. RIA and EIA are performed to study serum HLP and E levels throughout normal pregnancies. Results allow to use EIA for HLP and E evaluations in pregnancy-monitoring [fr

Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

DEFF Research Database (Denmark)

Hendriksen, Niels Bohse; Winding, Anne

2012-01-01

Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling...... of soil microbial functions is still needed. In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect...... the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose...

Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

International Nuclear Information System (INIS)

Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

1982-01-01

We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

OpenAIRE

Richardson, M D; Stubbins, J M; Warnock, D W

1982-01-01

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen ...

Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

Directory of Open Access Journals (Sweden)

Wiltrud Haaß

Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

Diagnosis of acute radiation disease by Enzyme Immune-Assay (EIA)

International Nuclear Information System (INIS)

Popov, D.; Maliev, V.; Jones, J.; Gonta, S.; Prasad, K.; Rachal, C.

2006-01-01

Diagnosis of the acute radiation disease by the method of immune enzyme assay is a simple and efficient tool of evaluating and biological dosimetry and forecasting of development of the acute radiation defeats as at group of population so at individuals locating in the zone polluted by the radiation. We use as biological markers the group of essential radiotoxins - high molecular mass glycoprotein ( molecular mass - 200 - 250 kDa ) - radiation antigens (S.D.R. - specific radiation determinant ) accumulated in the lymphoid system, with epitopes specific to each form of radiation syndrome, after animals have been irradiated in doses inducing the development of the cerebral (1), toxic ( 2), gastrointestinal ( 3 ) and typical ( 4 ) forms of acute radiation sickness. These two phenomena allowed us to develop a technologies for diagnosis, prophylaxis and therapy of radiation disease - enzyme immune assay ( EIA ), anti radiation vaccine, anti radiation serum, method of immune - lymph - plasma-sorption. The important first step in effectiveness of therapy is an accurate assessment of severity of disease in early period after irradiation. The ideal markers for early and accurate assessment is high weight glycoprotein with specifics radiation induced features (S.D.R.) mentioned above. This biology active substance isolated from lymph can induct the symptoms of radiation syndrome without previously radiation when it is administrated intra-muscularly or intravenously to healthy animals. Enzyme immune assay (EIA) allowed researchers to indicate the significant levels of different forms of S.D.R. in peripheral blood of animals in first 24 hours after radiation. Indication of high level of S.D.R. -1 allowed to forecast a fast development of cerebral form of acute radiation disease. Determination of high levels of S.D.R.-2, S.D.R.-3 and S.D.R.-4 in peripheral blood allowed to recognize early periods of toxic, gastrointestinal and typical forms of acute radiation sickness

Diagnosis of acute radiation disease by Enzyme Immune-Assay (EIA)

Energy Technology Data Exchange (ETDEWEB)

Popov, D.; Maliev, V. [Russian Academy of Science, Vladicaukas (Russian Federation); Jones, J.; Gonta, S. [NASA -Johnson Spa ce Center, Houston (United States); Prasad, K. [Antioxidant Research Institute, Premier Micrinutrient corporation, Novato (United States); Rachal, C. [Univercity Space Research Assotiation, Colorado (United States)

2006-07-01

Diagnosis of the acute radiation disease by the method of immune enzyme assay is a simple and efficient tool of evaluating and biological dosimetry and forecasting of development of the acute radiation defeats as at group of population so at individuals locating in the zone polluted by the radiation. We use as biological markers the group of essential radiotoxins - high molecular mass glycoprotein ( molecular mass - 200 - 250 kDa ) - radiation antigens (S.D.R. - specific radiation determinant ) accumulated in the lymphoid system, with epitopes specific to each form of radiation syndrome, after animals have been irradiated in doses inducing the development of the cerebral (1), toxic ( 2), gastrointestinal ( 3 ) and typical ( 4 ) forms of acute radiation sickness. These two phenomena allowed us to develop a technologies for diagnosis, prophylaxis and therapy of radiation disease - enzyme immune assay ( EIA ), anti radiation vaccine, anti radiation serum, method of immune - lymph - plasma-sorption. The important first step in effectiveness of therapy is an accurate assessment of severity of disease in early period after irradiation. The ideal markers for early and accurate assessment is high weight glycoprotein with specifics radiation induced features (S.D.R.) mentioned above. This biology active substance isolated from lymph can induct the symptoms of radiation syndrome without previously radiation when it is administrated intra-muscularly or intravenously to healthy animals. Enzyme immune assay (EIA) allowed researchers to indicate the significant levels of different forms of S.D.R. in peripheral blood of animals in first 24 hours after radiation. Indication of high level of S.D.R. -1 allowed to forecast a fast development of cerebral form of acute radiation disease. Determination of high levels of S.D.R.-2, S.D.R.-3 and S.D.R.-4 in peripheral blood allowed to recognize early periods of toxic, gastrointestinal and typical forms of acute radiation sickness

Use of laminar flow patterning for miniaturised biochemical assays

DEFF Research Database (Denmark)

Regenberg, Birgitte; Krühne, Ulrich; Beyer, M.

2004-01-01

Laminar flow in microfluidic chambers was used to construct low (one dimensional) density arrays suitable for miniaturized biochemical assays. By varying the ratio of flows of two guiding streams flanking a sample stream, precise focusing and positioning of the latter was achieved, and reactive s...... species carried in the sample stream were deposited on functionalized chip surfaces as discrete 50 mm wide lanes. Using different model systems we have confirmed the method's suitability for qualitative screening and quantification tasks in receptor-ligand assays, recording biotin...

Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay

DEFF Research Database (Denmark)

Jacobsen, Naja Wessel; Hansen, Cecilie Hurup; Nellemann, Christine

2015-01-01

shown to inhibit the aromatase enzyme in both types of aromatase assays. The IC50 values ranged from 3 to 600μM. All five SSRIs, were further investigated in the H295R cell line. All compounds altered the steroid secretion from the cells, the lowest observed effect levels were 0.9μM and 3.1μ....... In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were......M for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine...

Measurement of enzyme activity.

Science.gov (United States)

Harris, T K; Keshwani, M M

2009-01-01

To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

Real-time monitoring of enzyme-free strand displacement cascades by colorimetric assays

Science.gov (United States)

Duan, Ruixue; Wang, Boya; Hong, Fan; Zhang, Tianchi; Jia, Yongmei; Huang, Jiayu; Hakeem, Abdul; Liu, Nannan; Lou, Xiaoding; Xia, Fan

2015-03-01

The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications.The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications. Electronic supplementary information (ESI) available: Experimental procedures and analytical data are provided. See DOI: 10.1039/c5nr00697j

Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

Science.gov (United States)

Gurram, Raghu N; Menkhaus, Todd J

2014-07-01

Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products.

Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

Science.gov (United States)

Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

Desomorphine Screening Using Commercial Enzyme-Linked Immunosorbent Assays.

Science.gov (United States)

Winborn, Jessica; Kerrigan, Sarah

2017-06-01

Desomorphine ("Krokodil") is a semi-synthetic opioid that has drawn attention as a recreational drug, particularly in Russia, neighboring former Soviet Republics, Eastern and Central Europe. It has no accepted medicinal uses and is currently a schedule I drug in the United States. In clandestine environments, desomorphine is synthesized from codeine using red phosphorous, hydroiodic acid and gasoline. Residual starting materials in illicit preparations have been associated with severe dermatological effects and extensive tissue necrosis. Desomorphine is not well studied, and there are limited reports concerning its pharmacology or detection in biological matrices. Immunoassays are widely relied upon for both antemortem and postmortem toxicology screening. Although desomorphine is an opioid of the phenanthrene-type, its ability to bind to conventional opioid antibodies has not been described. In this report we describe the cross-reactivity of desomorphine using six commercially available enzyme-linked immunosorbent assays (Immunalysis Opiates Direct ELISA, Immunalysis Oxycodone/Oxymorphone Direct ELISA, Randox Opiate ELISA, OraSure Technologies OTI Opiate Micro-plate EIA, Neogen Opiate Group ELISA and Neogen Oxycodone/Oxymorphone ELISA). Cross-reactivites were highly variable between assays, ranging from 77 to desomorphine than those directed towards oxycodone. The Immunalysis Opiates Direct ELISA produced the greatest cross-reactivity, although several of the assays evaluated produced cross-reactivity of a sufficient magnitude to be effective for desomorphine screening. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

DEFF Research Database (Denmark)

Rasmussen, Randi Engelberth; Erstad, Simon Matthé; Ramos Martinez, Erick Miguel

2016-01-01

microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls...... used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process...... for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism...

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

Science.gov (United States)

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

α-amylase assay and action pattern determination using radioactive substrate, HPLC, and a radioactive flow detector

International Nuclear Information System (INIS)

Marsili, R.T.; Ostapenko, H.

1987-01-01

A new assay system is presented for the analysis of α-amylase. The disappearance of 14 C-labeled starch substrate and the appearance of its radioactive degradation products were monitored by HPLC and a radioactive flow detector/integrator. The hydrolysis of radioactive substrate was proportional to enzyme concentration when two commercially available α-amylase preparations of Bacillus subtilis origin were studied. The method demonstrated an average recovery of 101.7 +/- 6.5% when modified food starch was spiked with amylase and analyzed. In addition, the method was shown to be useful for predicting detailed action patterns of various types of amylases

The relevance of chemical interactions with CYP17 enzyme activity: Assessment using a novel in vitro assay

International Nuclear Information System (INIS)

Roelofs, Maarke J.E.; Piersma, Aldert H.; Berg, Martin van den; Duursen, Majorie B.M. van

2013-01-01

The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. - Highlights: ► DHEA, produced by CYP17, plays a key role in sexual functioning and development. ► No rapid screening assay for effects on CYP17 is available yet. ► A novel assay using porcine adrenal cortex microsomes (PACMs) was described. ► Endocrine disrupting compounds (EDCs) targeting CYP17 interact differently with CYP19. ► A

The relevance of chemical interactions with CYP17 enzyme activity: Assessment using a novel in vitro assay

Energy Technology Data Exchange (ETDEWEB)

Roelofs, Maarke J.E., E-mail: m.j.e.roelofs@uu.nl [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands); Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Piersma, Aldert H. [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands); Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Berg, Martin van den; Duursen, Majorie B.M. van [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands)

2013-05-01

The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. - Highlights: ► DHEA, produced by CYP17, plays a key role in sexual functioning and development. ► No rapid screening assay for effects on CYP17 is available yet. ► A novel assay using porcine adrenal cortex microsomes (PACMs) was described. ► Endocrine disrupting compounds (EDCs) targeting CYP17 interact differently with CYP19. ► A

Detection of thyroid stimulating hormone receptor antibodies (TRAb) by radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA)

International Nuclear Information System (INIS)

Dumrongpisutikul, S.; Tuchinda, S.

1990-01-01

Thyroid stimulating hormone receptor antibodies (TRAb) were determined in 100 patients using radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of RRA and ELISA were found to be 70.6% and 88.2% respectively (n=51). The specificity of both assays were 100% (n=16). With RRA as the standard test the sensitivity and specificity of ELISA were 75.8% and 86.8%. In the untreated hyperthyroid the RRA result which expressed as % specific 125 I-TSH inhibition was 33.6% (n=51), decline to 26.9% in the treated hyperthyroid (n=33) and 14.1% in the euthyroid (n=16). The mean 0.D 492nm of TRAb-ELISA were 0.861 in untreated hyperthyroid, 0.437 in treated hyperthyroid and 0.135 in euthyroid Phi coefficient analysis show that the RRA was 60.4% correlated to hyperthyroidism where as TRAb-ELISA was 80.1%

Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins.

Science.gov (United States)

Carmichael, W W; An, J

1999-01-01

Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

Science.gov (United States)

We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

Effect of bilirubin on the spectrophotometric and radionuclide assay for serum angiotensin-converting enzyme

International Nuclear Information System (INIS)

Saxe, A.W.; Hollinger, M.A.; Essam, T.

1986-01-01

The effect of bilirubin on serum angiotensin-converting enzyme (ACE) activity was studied with spectrophotometric and radionuclide assays. In the spectrophotometric assay addition of bilirubin to normal serum from dog, mouse, and human produced a dose-related inhibition of ACE activity. A 50% decrease in human ACE activity was produced by the addition of approximately 250 mg/L in vitro. Serum from icteric patients with elevated bilirubin was also associated with a reduction in ACE activity in the spectrophotometric assay. A 50% decrease in ACE activity in these samples was associated with a serum bilirubin of approximately 220 mg/L. In the radionuclide assay, however, addition of bilirubin to normal human serum failed to reduce measured ACE activity. The use of a radionuclide assay for serum ACE in clinical samples offers the advantage of less interference from serum bilirubin

rK39 enzyme-linked immunosorbent assay for diagnosis of Leishmania donovani infection

NARCIS (Netherlands)

Zijlstra, E. E.; Daifalla, N. S.; Kager, P. A.; Khalil, E. A.; El-Hassan, A. M.; Reed, S. G.; Ghalib, H. W.

1998-01-01

The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months

DYNAMIC MODELLING AND ADVANCED PREDICTIVE CONTROL OF A CONTINUOUS PROCESS OF ENZYME PURIFICATION

Directory of Open Access Journals (Sweden)

Dechechi E.C.

1997-01-01

Full Text Available A dynamic mathematical model, simulation and computer control of a Continuous Affinity Recycle Extraction (CARE process, a protein purification technique based on protein adsorption on solid-phase adsorbents is described in this work. This process, consisting of three reactors, is a multivariable process with considerable time delay in the on-line analyses of the controlled variable. An advanced predictive control configuration, specifically the Dynamic Matrix Control (DMC, was applied. The DMC algorithm was applied in process schemes where the aim was to maintain constant the enzyme concentration in the outlet of the third reactor. The performance of the DMC controller was analyzed in the feed-flow disturbances and the results are presented.

Quantification of methanogenic biomass by enzyme-linked immunosorbent assay and by analysis of specific methanogenic cofactors

Energy Technology Data Exchange (ETDEWEB)

Gorris, L G.M.; Kemp, H A; Archer, D B

1987-01-01

The reliability and accuracy with which enzyme-linked immunosorbent assay (ELISA) and an assay of methanogenic cofactors detect and quantify methanogenic species were investigated. Both assays required standardization with laboratory cultures of methanogenic bacteria and were applied to mixtures of pure cultures and samples from anaerobic digesters. ELISA was shown to be a simple method for detecting and quantifying individual methanogenic species. The range of species which can be assayed is limited by the range of antisera available but, potentially, ELISA can be applied to all methanogens. Although the cofactor assay is not species-specific it can distinguish hydrogenotrophic and acetotrophic methanogens and is quantitative.

Immunodiagnosis of Human Fascioliasis by an Enzyme-Linked Immunosorbent Assay (ELISA) and a Micro-ELISA

OpenAIRE

Carnevale, Silvana; Rodríguez, Mónica I.; Santillán, Graciela; Labbé, Jorge H.; Cabrera, Marta G.; Bellegarde, Enrique J.; Velásquez, Jorge N.; Trgovcic, Jorge E.; Guarnera, Eduardo A.

2001-01-01

Enzyme-linked immunosorbent assay (ELISA) and micro-ELISA were evaluated for their ability to detect anti-Fasciola hepatica antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis.

A homogeneous assay principle for universal substrate quantification via hydrogen peroxide producing enzymes

International Nuclear Information System (INIS)

Zscharnack, Kristin; Kreisig, Thomas; Prasse, Agneta A.; Zuchner, Thole

2015-01-01

Highlights: • Application of the TRF-based PATb system for universal oxidase substrate detection. • H 2 O 2 generated by choline or glucose oxidase quenches the TRF signal of PATb. • The assay time is only limited by the oxidase catalysis rate. • Glucose is precisely detected in human serum consistent to a commercial assay. • A reliable quantification of choline in infant formula is shown. - Abstract: H 2 O 2 is a widely occurring molecule which is also a byproduct of a number of enzymatic reactions. It can therefore be used to quantify the corresponding enzymatic substrates. In this study, the time-resolved fluorescence emission of a previously described complex consisting of phthalic acid and terbium (III) ions (PATb) is used for H 2 O 2 detection. In detail, glucose oxidase and choline oxidase convert glucose and choline, respectively, to generate H 2 O 2 which acts as a quencher for the PATb complex. The response time of the PATb complex toward H 2 O 2 is immediate and the assay time only depends on the conversion rate of the enzymes involved. The PATb assay quantifies glucose in a linear range of 0.02–10 mmol L −1 , and choline from 1.56 to 100 μmol L −1 with a detection limit of 20 μmol L −1 for glucose and 1.56 μmol L −1 for choline. Both biomolecules glucose and choline could be detected without pretreatment with good precision and reproducibility in human serum samples and infant formula, respectively. Furthermore, it is shown that the detected glucose concentrations by the PATb system agree with the results of a commercially available assay. In principle, the PATb system is a universal and versatile tool for the quantification of any substrate and enzyme reaction where H 2 O 2 is involved

Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale

International Nuclear Information System (INIS)

Haven, Mai Østergaard; Lindedam, Jane; Jeppesen, Martin Dan; Elleskov, Michael; Rodrigues, Ana Cristina; Gama, Miguel; Jørgensen, Henning; Felby, Claus

2015-01-01

Highlights: • Results from continuous experiments in demonstration scale for a total of 16 days. • Reuse of enzymes is possible through recycling fermentation broth. • Recycling fermentation broth can increase ethanol concentration with lower dry matter. - Abstract: Recycling of enzymes in production of lignocellulosic bioethanol has been tried for more than 30 years. So far, the successes have been few and the experiments have been carried out at conditions far from those in an industrially feasible process. Here we have tested continuous enzyme recycling at demonstration scale using industrial process conditions (high dry matter content and low enzyme dosage) for a period of eight days. The experiment was performed at the Inbicon demonstration plant (Kalundborg, Denmark) capable of converting four tonnes of wheat straw per hour. 20% of the fermentation broth was recycled to the hydrolysis reactor while enzyme dosage was reduced by 5%. The results demonstrate that recycling enzymes by this method can reduce overall enzyme consumption and may also increase the ethanol concentrations in the fermentation broth. Our results further show that recycling fermentation broth also opens up the possibility of lowering the dry matter content in hydrolysis and fermentation while still maintaining high ethanol concentrations.

Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

Science.gov (United States)

Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

2014-01-01

Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

Designs, formats and applications of lateral flow assay: A literature review

Directory of Open Access Journals (Sweden)

Muhammad Sajid

2015-11-01

Full Text Available This manuscript provides a brief overview of latest research involving the use of lateral flow assay for qualitative and quantitative analysis in different areas. The excellent features and versatility of detection formats make these strips an ideal choice for point of care applications. We outline and critically discuss detection formats, molecular recognition probes, labels, and detection systems used in lateral flow assay. Applications in different fields along with selected examples from the literature have been included to show analytical performance of these devices. At the end, we summarize accomplishments, weaknesses and future challenges in the area of lateral flow strips.

Comparative study of the enzyme linked immunosorbent assay (ELISA) and two radioimmunoassays (RIA'S) for in-sulin

Energy Technology Data Exchange (ETDEWEB)

Klimes, I; Jurcovicova, J; Palkovic, M [Slovenska Akademia Vied, Bratislava (Czechoslovakia). Ustav Experimentalnej Endokrinologie

1978-06-30

The results of the quality control tests for enzyme linked immunosorbent assay (ELISA) were compared with the results of two different radioimmunoassays (RIA'S) for insulin. Using the manufacturer's procedure for the ELISA kit we found that the analytical variables such as assay sensitivity, recovery study and the 50% binding intercept were in good agreement with those obtained with the RIA method.

Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants.

Science.gov (United States)

Smirnova, Julia; Fernie, Alisdair R; Spahn, Christian M T; Steup, Martin

2017-09-01

Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and β-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify β-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. Copyright © 2017. Published by Elsevier Inc.

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

Science.gov (United States)

SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

2013-01-01

ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

Diagnosis of selected tropical diseases (Shistosomiasis and Malaria) using the enzyme-linked immunosorbent assays

International Nuclear Information System (INIS)

Chembe, E.

1985-01-01

Immunological reactions are commonly used in diagnostic procedures on the basis of their high levels of specificity and sensitivity. Antibodies or antigens labelled with various markers have been found to be particularly useful for assays of logical substances. The applications of Enzyme-Linked Immunoabsorbent Assays (ELISA) to research on various tropical and non-tropical diseases is now well established. The procedure depends on the labelling of one of the reactants with enzymes which can be detected accurately by an appropriate substrate. The detection mechanism depends on the labelling of one of the reactants in such a way that their their reactivity is not impaired or affected. In the present study, ELISA was applied to sera from kampumbu area of Isoka district in the Northern province of Zambia. The objective of this presentation is to show the relative positivity rate for antigen and antibody and the endemicity of schistosomiasis and malaria as assessed by classical parasitological procedures. (author)

Liquid phase oxidation chemistry in continuous-flow microreactors.

Science.gov (United States)

Gemoets, Hannes P L; Su, Yuanhai; Shang, Minjing; Hessel, Volker; Luque, Rafael; Noël, Timothy

2016-01-07

Continuous-flow liquid phase oxidation chemistry in microreactors receives a lot of attention as the reactor provides enhanced heat and mass transfer characteristics, safe use of hazardous oxidants, high interfacial areas, and scale-up potential. In this review, an up-to-date overview of both technological and chemical aspects of liquid phase oxidation chemistry in continuous-flow microreactors is given. A description of mass and heat transfer phenomena is provided and fundamental principles are deduced which can be used to make a judicious choice for a suitable reactor. In addition, the safety aspects of continuous-flow technology are discussed. Next, oxidation chemistry in flow is discussed, including the use of oxygen, hydrogen peroxide, ozone and other oxidants in flow. Finally, the scale-up potential for continuous-flow reactors is described.

Continuous flow nitration in miniaturized devices

Directory of Open Access Journals (Sweden)

Amol A. Kulkarni

2014-02-01

Full Text Available This review highlights the state of the art in the field of continuous flow nitration with miniaturized devices. Although nitration has been one of the oldest and most important unit reactions, the advent of miniaturized devices has paved the way for new opportunities to reconsider the conventional approach for exothermic and selectivity sensitive nitration reactions. Four different approaches to flow nitration with microreactors are presented herein and discussed in view of their advantages, limitations and applicability of the information towards scale-up. Selected recent patents that disclose scale-up methodologies for continuous flow nitration are also briefly reviewed.

ANALYSIS OF SOIL AND DUST SAMPLES FOR POLYCHLORINATED BIPHENYLS BY ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

Science.gov (United States)

An inhibition enzyme-linked immunosorbent assay (ELISA) was used to determine polychlorinated biphenyls (PCBs) in house dust and soil. Soil and house dust samples were analyzed for PCB by both gas chromatography/electron capture detection (GC/ECD) and ELISA methods. A correlati...

Phytochrome quantitation in crude extracts of Avena by enzyme-linked immunosorbent assay with monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Shimazaki, Y; Cordonnier, M M; Pratt, L H

1983-01-01

An enzyme-linked immunosorbent assay (ELISA), which uses both rabbit polyclonal and mouse monoclonal antibodies to phytochrome, has been adapted for quantitation of phytochrome in crude plant extracts. The assay has a detection limit of about 100 pg phytochrome and can be completed within 10 h. Quantitation of phytochrome in crude extracts of etiolated oat seedlings by ELISA gave values that agreed well with those obtained by spectrophotometric assay. When etiolated oat seedlings were irradiated continuously for 24 h, the amount of phytochrome detected by ELISA and by spectrophotometric assay decreased by more than 1000-fold and about 100-fold, respectively. This discrepancy indicates that phytochrome in light-treated plants may be antigenically distinct from that found in fully etiolated plants. When these light-grown oat seedlings were kept in darkness for 48 h, phytochrome content detected by ELISA increased by 50-fold in crude extracts of green oat shoots, but only about 12-fold in extracts of herbicide-treated oat shoots. Phytochrome reaccumulation in green oat shoots was initially more rapid in the more mature cells of the primary leaf tip than near the basal part of the shoot. The inhibitory effect of Norflurazon on phytochrome accumulation was much more evident near the leaf tip than the shoot base. A 5-min red irradiation of oat seedlings at the end of a 48-h dark period resulted in a subsequent, massive decrease in phytochrome content in crude extracts from both green and Norflurazon-bleached oat shoots. These observations eliminate the possibility that substantial accumulation of chromophore-free phytochrome was being detected and indicate that Norflurazon has a substantial effect on phytochrome accumulation during a prolonged dark period. 25 references, 9 figures, 3 tables.

A homogeneous assay principle for universal substrate quantification via hydrogen peroxide producing enzymes

Energy Technology Data Exchange (ETDEWEB)

Zscharnack, Kristin; Kreisig, Thomas; Prasse, Agneta A. [Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig (Germany); Zuchner, Thole, E-mail: Thole.Zuechner@octapharma.com [Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig (Germany); Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig (Germany)

2015-01-07

Highlights: • Application of the TRF-based PATb system for universal oxidase substrate detection. • H{sub 2}O{sub 2} generated by choline or glucose oxidase quenches the TRF signal of PATb. • The assay time is only limited by the oxidase catalysis rate. • Glucose is precisely detected in human serum consistent to a commercial assay. • A reliable quantification of choline in infant formula is shown. - Abstract: H{sub 2}O{sub 2} is a widely occurring molecule which is also a byproduct of a number of enzymatic reactions. It can therefore be used to quantify the corresponding enzymatic substrates. In this study, the time-resolved fluorescence emission of a previously described complex consisting of phthalic acid and terbium (III) ions (PATb) is used for H{sub 2}O{sub 2} detection. In detail, glucose oxidase and choline oxidase convert glucose and choline, respectively, to generate H{sub 2}O{sub 2} which acts as a quencher for the PATb complex. The response time of the PATb complex toward H{sub 2}O{sub 2} is immediate and the assay time only depends on the conversion rate of the enzymes involved. The PATb assay quantifies glucose in a linear range of 0.02–10 mmol L{sup −1}, and choline from 1.56 to 100 μmol L{sup −1} with a detection limit of 20 μmol L{sup −1} for glucose and 1.56 μmol L{sup −1} for choline. Both biomolecules glucose and choline could be detected without pretreatment with good precision and reproducibility in human serum samples and infant formula, respectively. Furthermore, it is shown that the detected glucose concentrations by the PATb system agree with the results of a commercially available assay. In principle, the PATb system is a universal and versatile tool for the quantification of any substrate and enzyme reaction where H{sub 2}O{sub 2} is involved.

Assay of repair enzyme activity by reactivation of ultraviolet-irradiated infective viral DNA

Energy Technology Data Exchange (ETDEWEB)

Oeda, K; Nakatsu, Y; Sekiguchi, M [Kyushu Univ., Fukuoka (Japan).Faculty of Science

1980-05-01

Treatment of OeX174 replicative form (RF) DNA, pre-exposed to ultraviolet light, with T4 endonuclease V led to a marked increase of infectivity of the RF when the activity was assayed on CaCl/sub 2/-treated cells of Escherichia coli strain defective in uvrA gene. The reaction was specific and the extent of the reactivation was proportional to the concentration of the enzyme. Based on this finding, we developed a procedure to assay endonuclease activities specific for ultraviolet-damaged DNA, that might be involved in the incision step of excision repair of pyrimidine dimers. To find conditions suitable for accurate and rapid assays, we examined conditions affecting transfection with OeX174 RF. The maximum transfection was achieved when more than 2 x 10/sup 8/ CaCl/sub 2/-treated cells, which had been prepared from bacteria harvested during the early or mid-logarithmic phase of growth in L broth, were incubated with the DNA at 0/sup 0/C for 20 min in 50 mM CaCl/sub 2/. Incubation of the cell-DNA mixture at 37/sup 0/C decreased the transfection efficiency to about 30% of the optimal level; thus, heat shock, a step regarded as necessary in the conventional CaCl/sub 2/ methods for transfection and transformation, was eliminated. The CaCl/sub 2/-treated cells remained viable and competent after storage at -20/sup 0/C in a solution containing 15% glycerol. By using the procedure thus established, repair endonuclease activities in crude extracts of T4-infected E. coli and of Micrococcus luteus were determined. The procedure should be of use in assaying and purifying repair enzymes of other organisms.

Application of an improved enzyme-linked immunosorbent assay method for serological diagnosis of canine leishmaniasis

NARCIS (Netherlands)

Santarém, Nuno; Silvestre, Ricardo; Cardoso, Luís; Schallig, Henk; Reed, Steven G.; Cordeiro-da-Silva, Anabela

2010-01-01

Accurate diagnosis of canine leishmaniasis (CanL) is essential toward a more efficient control of this zoonosis, but it remains problematic due to the high incidence of asymptomatic infections. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based

Mobile magnetic particles as solid-supports for rapid surface-based bioanalysis in continuous flow.

Science.gov (United States)

Peyman, Sally A; Iles, Alexander; Pamme, Nicole

2009-11-07

An extremely versatile microfluidic device is demonstrated in which multi-step (bio)chemical procedures can be performed in continuous flow. The system operates by generating several co-laminar flow streams, which contain reagents for specific (bio)reactions across a rectangular reaction chamber. Functionalized magnetic microparticles are employed as mobile solid-supports and are pulled from one side of the reaction chamber to the other by use of an external magnetic field. As the particles traverse the co-laminar reagent streams, binding and washing steps are performed on their surface in one operation in continuous flow. The applicability of the platform was first demonstrated by performing a proof-of-principle binding assay between streptavidin coated magnetic particles and biotin in free solution with a limit of detection of 20 ng mL(-1) of free biotin. The system was then applied to a mouse IgG sandwich immunoassay as a first example of a process involving two binding steps and two washing steps, all performed within 60 s, a fraction of the time required for conventional testing.

Performance evaluation of FlowCytomix assays to quantify cytokines in patients with rheumatoid arthritis

Science.gov (United States)

Wang, Xuefeng; Dong, Liyang; Liang, Yong; Ni, Hongchang; Tang, Jun; Xu, Chengcheng; Zhou, Yuepeng; Su, Yuting; Wang, Jun; Chen, Deyu; Mao, Chaoming

2015-01-01

Objectives: To compare the cytokine profile in RA patients and healthy control by using two methods-FlowCytomix assay and traditional ELISA. Methods: Cytokine levels were evaluated by FlowCytomix assay and ELISA in serum and supernatants of peripheral blood mononuclear cells (PBMC) cultures with and without stimulation by phytohaemagglutinin (PHA). Results: The levels of IL-6, IL-1β, and TNF-α were significantly higher in sera of RA patients than those of healthy controls. The levels of IL-22, IL-6, IL-1β, TNF-α, and IL-10 were higher in unstimulated PBMC culture supernatant of RA patients than those of healthy controls. PHA stimulation significantly increased the production of proinflammatory cytokines from PBMC with RA patients. Compared with detectable cytokine levels in sera, cytokine concentration in the supernatant of PBMCs was remarkably higher. FlowCytomix and ELISA showed significant correlation in detecting cytokines. However, the FlowCytomix assay detected more cytokines than ELISA. Conclusion: The supernatant of PBMCs provide a fine condition for the study of cytokine production because of the lack of interference factors in sera. The FlowCytomix assay is more sensitive than ELISA in detecting cytokines from RA patients. Multiple cytokine signatures using FlowCytomix assay may represent a more realistic approach in the future of personalized medicine in RA. PMID:26629129

A flow cytometric assay for simultaneously measuring the ...

African Journals Online (AJOL)

This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

An Inhibitive Enzyme Assay to Detect Mercury and Zinc Using Protease from Coriandrum sativum

Directory of Open Access Journals (Sweden)

Gunasekaran Baskaran

2013-01-01

Full Text Available Heavy metals pollution has become a great threat to the world. Since instrumental methods are expensive and need skilled technician, a simple and fast method is needed to determine the presence of heavy metals in the environment. In this study, an inhibitive enzyme assay for heavy metals has been developed using crude proteases from Coriandrum sativum. In this assay, casein was used as a substrate and Coomassie dye was used to denote the completion of casein hydrolysis. In the absence of inhibitors, casein was hydrolysed and the solution became brown, while in the presence of metal ions such as Hg2+ and Zn2+, the hydrolysis of casein was inhibited and the solution remained blue. Both Hg2+ and Zn2+ exhibited one-phase binding curve with IC50 values of 3.217 mg/L and 0.727 mg/L, respectively. The limits of detection (LOD and limits of quantitation (LOQ for Hg were 0.241 and 0.802 mg/L, respectively, while the LOD and LOQ for Zn were 0.228 and 0.761 mg/L, respectively. The enzyme exhibited broad pH ranges for activity. The crude proteases extracted from Coriandrum sativum showed good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of Hg2+ and Zn2+ in the aquatic environments.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.

Science.gov (United States)

Ding, Wei; Bishop, Michelle E; Lyn-Cook, Lascelles E; Davis, Kelly J; Manjanatha, Mugimane G

2016-05-04

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.

Science.gov (United States)

Zhu, Linling; Zhang, Junying; Wang, Fengyang; Wang, Ya; Lu, Linlin; Feng, Chongchong; Xu, Zhiai; Zhang, Wen

2016-04-15

Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli.

Science.gov (United States)

Birnbaum, S; Bülow, L; Hardy, K; Danielsson, B; Mosbach, K

1986-10-01

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.

A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

Science.gov (United States)

Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

2012-01-01

ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

DEFF Research Database (Denmark)

Wiggers, Henrik; Cheleski, J; Zottis, A

2007-01-01

.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity......In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected...... by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (Me...

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein

DEFF Research Database (Denmark)

Rasmussen, M; Dahl, M; Buus, S

2014-01-01

. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide...... as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed....../ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer...

EVALUATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR BIOLOGICAL MONITORING OF 3-PHENOXYBENZOIC ACID IN URINE

Science.gov (United States)

Abstract describes the development of an enzyme-linked immunosorbent assay (ELISA) method for monitoring 2,4-dichlorophenoxyacetic acid (2,4-D exposures). The ELISA is compared with a gas chromatograhy/mass spectrometry procedure. ELISA method development steps and comparative ...

Validation of 2 commercially available enzyme-linked immunosorbent assays for adiponectin determination in canine serum samples.

Science.gov (United States)

Tvarijonaviciute, Asta; Martínez-Subiela, Silvia; Ceron, José J

2010-10-01

The aim of this study was to validate 2 commercially available enzyme-linked immunosorbent assays (ELISAs) for adiponectin in dogs, 1 canine-specific and 1 originally designed for measurements in humans. Intra-assay and interassay precision was evaluated by multiple measurements in canine serum samples, and assay accuracy was indirectly determined by linearity under dilution. Interference caused by hemolysis and lipemia was also studied. Both assays were subsequently used for measuring adiponectin concentrations in clinically healthy dogs and those with different grades of obesity. The intra-assay and inter-assay precision was less than 7.5% and 13.5% in serum samples with low and high adiponectin concentrations, respectively. Lipemia and hemolysis did not affect the results of any of the assays. Both assays were able to differentiate lean dogs from those that were overweight or obese on the basis of the measured adiponectin concentrations. From these results it can be concluded that canine adiponectin concentrations can be measured reliably by means of the 2 ELISAs evaluated in this study.

A simple assay method for omega-oxidation of lauric acid by hepatic enzymes

International Nuclear Information System (INIS)

Giera, D.D.; van Lier, R.B.L.

1990-01-01

Routine assessment of hepatic ω-oxidation of fatty acids in toxicology studies requires a simpler method of enzymatic analysis than HPLC or TLC. A method depending upon selective solvent separation of 14 C-lauric acid and 14 C-11/12-hydroxy lauric acid was developed. Following enzymatic incubation and addition of 15% methanol to the acidified incubation mixtures, partitioning with an alkane solvent such as iso-octane, cyclohexane, or n-hexane separated the lauric acid substrate and ω-hydroxylated products into two immiscible phases. Approximately 98% of the substrate partitioned into the organic phase, and approximately 83% of the hydroxylated products partitioned into the aqueous phase. Subsequent quantitation of the enzymatic activity required only liquid scintillation counting of the aqueous phase. Hepatic homogenates from male rats treated with 0.01, 0.05, 0.125, and 0.25% clofibrate in the diet for 7 days had enzyme levels 1.3, 6.1, 11.1, and 15.9 times control values, respectively, when assayed by conventional TLC methods, and 1.3, 5.3, 12.3, and 15.3 times control values when assayed by the solvent extraction method. The data indicate that the selective solvent partitioning yields comparable precision and sensitivity to the more conventional TLC method when studying induction of hepatic microsomal enzymes

Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

Science.gov (United States)

Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

2010-05-26

Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays

Directory of Open Access Journals (Sweden)

Helen V. Hsieh

2017-05-01

Full Text Available Immunochromatographic or lateral flow assays (LFAs are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor’s office in Manhattan to a rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads, biological reagents (e.g., antibodies, blocking reagents and buffers and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness.

Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

Science.gov (United States)

Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-06-07

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode.

Science.gov (United States)

Hernández-Neuta, Iván; Pereiro, Iago; Ahlford, Annika; Ferraro, Davide; Zhang, Qiongdi; Viovy, Jean-Louis; Descroix, Stéphanie; Nilsson, Mats

2018-04-15

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120μL of DNA dilution at flow rates ranging from 1 to 5μL/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

A Calorimetric Assay For Enzymatic Saccharification Of Biomass

DEFF Research Database (Denmark)

Murphy, Leigh; Borch, Kim; McFarland, K.C.

2010-01-01

A limited selection of assay and screening methodologies for cellulolytic enzymes has been stated as a restriction in biomass research. In this report we test the potential of isothermal calorimetry for this purpose. The primary observable in this technique (the heat flow in Watts), scales with t...... of the regulation and functional mechanism of cellulases....

Cathepsin D immobilized capillary reactors for on-flow screening assays.

Science.gov (United States)

Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

2018-03-20

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli

International Nuclear Information System (INIS)

Birnbaum, S.; Buelow, L.; Hardy, K.; Danielsson, B.; Mosbach, K.

1986-01-01

The authors have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 μg/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 0 C in the enzyme thermistor unit. Thus, immediate assay start up was possible

Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

2013-03-01

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

Science.gov (United States)

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Diagnosis of acute radiation disease by enzyme immune-assay (EIA)

International Nuclear Information System (INIS)

Popov, D.; Maliev, V.

2005-01-01

Full text: Multifactor fundamental research under experimental and industrial conditions on farm and laboratory animals and also on humans blood serum of Chernobyl NPP clean-up workers conducted within 1982 - 2002 have enabled us to establish the existence of two previously unknown phenomena: the phenomenon of reversible redistribution of cytobiochemical parameters in the blood-interstices-lymph-blood system of irradiated animals, which supports compensatory maintenance of homeostasis. The phenomenon of specific immunochemical reactions to the radiological effect, involving the formation of high molecular mass glycoprotein (molecular mass - 200 - 250 kDa) - radiation antigens (SDR - specific radiation determinant) in the lymphoid system with epitopes specific to each form of radiation syndrome, after animals have been irradiated in doses inducing the development of the cerebral (1) , toxic (2) gastrointestinal (3) and typical (4) forms of acute radiation sickness. These two phenomena allowed us to develop a technologies for diagnosis, prophylaxis and therapy of radiation disease - enzyme immune assay (EIA), antiradiation vaccine, antiradiation serum, method of immune-lymph-plasmosorption. The important first step in effectiveness of therapy is an accurate assessment of severity of disease in early period after irradiation. The ideal markers for early and accurate assessment is high weight glycoprotein with specifics radiation induced features (SDR) mentioned above. This biology active substance isolated from lymph can induct the symptoms of radiation syndrome without previously radiation when it is administrated intramuscularly or intravenously to healthy animals. Enzyme immune assay (EIA) allowed researchers to indicate the significant levels of different forms of SDR in peripheral blood of animals in first 24 hours after radiation. Indication of high level of SDR -1 allowed to forecast a fast development of cerebral form of acute radiation disease. Determination

On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes.

Science.gov (United States)

Maier, Manfred; Radtke, Carsten P; Hubbuch, Jürgen; Niemeyer, Christof M; Rabe, Kersten S

2018-05-04

The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three-dimensional (3D) printing techniques offer high-throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose-based inks for a simple temperature-controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D-printed parts for a continuous, two-step sequential biotransformation in a fluidic setup. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

Science.gov (United States)

Ackerman, S B; Kelley, E A

1983-03-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

Science.gov (United States)

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Energy Technology Data Exchange (ETDEWEB)

Cossar, D; Canevascini, G

1986-07-01

The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellubiose dehydrogenase remain to be elucidated.

Development of an enzyme-linked immunosorbent assay for the detection of ciguatoxin in fish tissue using chicken immunoglobulin Y.

Science.gov (United States)

Empey Campora, Cara; Hokama, Yoshitsugi; Yabusaki, Kenichi; Isobe, Minoru

2008-01-01

A sandwich enzyme-linked immunosorbent assay was developed to detect ciguatoxin (CTX) in fish tissue. The assay utilizes two antibodies, chicken immunoglobulin Y specific to the ABCD domain of CTX and a mouse monoclonal immunoglobulin G-horseradish peroxidase conjugate specific to the JKLM domain of CTX. The sensitivity, working range, cross reactivity, accuracy, precision, and reproducibility were examined.

Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

Science.gov (United States)

Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

2018-06-26

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

Science.gov (United States)

Jacobson, R H; Downing, D R; Lynch, T J

1982-11-15

A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

Preparation of nanoparticles by continuous-flow microfluidics

International Nuclear Information System (INIS)

Jahn, Andreas; Reiner, Joseph E.; Vreeland, Wyatt N.; DeVoe, Don L.; Locascio, Laurie E.; Gaitan, Michael

2008-01-01

We review a variety of micro- and nanoparticle formulations produced with microfluidic methods. A diverse variety of approaches to generate microscale and nanoscale particles has been reported. Here we emphasize the use of microfluidics, specifically microfluidic systems that operate in a continuous flow mode, thereby allowing continuous generation of desired particle formulations. The generation of semiconductor quantum dots, metal colloids, emulsions, and liposomes is considered. To emphasize the potential benefits of the continuous-flow microfluidic methodology for nanoparticle generation, preliminary data on the size distribution of liposomes formed using the microfluidic approach is compared to the traditional bulk alcohol injection method.

Two new automated, compared with two enzyme-linked immunosorbent, antimüllerian hormone assays.

Science.gov (United States)

Nelson, Scott M; Pastuszek, Ewa; Kloss, Grzegorz; Malinowska, Iwona; Liss, Joanna; Lukaszuk, Aron; Plociennik, Lukasz; Lukaszuk, Krzysztof

2015-10-01

To compare new automated antimüllerian hormone (AMH) assay performance characteristics from the new automated Elecsys AMH (Roche; Elecsys) and Access AMH (Beckman Coulter; Access) assays with the existing AMH Gen II ELISA (enzyme-linked immunosorbent assay; Gen II; Beckman Coulter) and AMH ELISA (Ansh Labs) assays. Prospective assay evaluation. University-affiliated clinical chemistry laboratory. Patients referred for serum AMH measurement (n = 83) before start of in vitro fertilization cycle between September 2014 and October 2014. None. Serum AMH concentration. Intra-assay coefficients of variation were low; Ansh ≤ 9.0%; Gen II ≤ 5.8%; Access ≤ 10.7%; and Elecsys ≤ 2.8%. The Passing-Bablok regression equations (pmol/L) were y (Access) = 0.128 + (0.781 × Gen II); and y (Access) = 0.302 + (0.742 x Ansh). For y (Elecys) = 0.087 + (0.729 x Gen II) and y (Elecys) = 0.253 + (0.688 x Ansh Labs). For y (Elecys) = 0.943 - (0.037 × Access). For all the assays, AMH exhibited a moderate positive correlation with AFC (r = 0.62-0.64); number of cumulus oocyte complexes (r = 0.60-0.64); and metaphase II oocytes (r = 0.48-0.50). Accuracy of pregnancy prediction, as determined by area under the receiver operating characteristic curve, was uniformly low for all assays (0.62-0.63). The novel automated assays exhibit strong concordance in calibration, but derived values are substantially lower than those obtained from pre-existing assays, with assay-specific interpretation required for routine clinical use. These results highlight the need for an international standard of measurement of AMH. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Catalytic Synthesis of Nitriles in Continuous Flow

DEFF Research Database (Denmark)

Nordvang, Emily Catherine

The objective of this thesis is to report the development of a new, alternative process for the flexible production of nitrile compounds in continuous flow. Nitriles are an important class of compounds that find applications as solvents, chemical intermediates and pharmaceutical compounds......, alternative path to acetonitrile from ethanol via the oxidative dehydrogenation of ethylamine. The catalytic activity and product ratios of the batch and continuous flow reactions are compared and the effect of reaction conditions on the reaction is investigated. The effects of ammonia in the reaction...... dehydrogenation of ethylamine and post-reaction purging.Chapter 4 outlines the application of RuO2/Al2O3 catalysts to the oxidative dehydrogenation of benzylamine in air, utilizing a new reaction setup. Again, batch and continuous flow reactions are compared and the effects of reaction conditions, ammonia...

Enzyme-linked immunosorbent assay characterization of Basal variation and heritability of systemic microfibrillar-associated protein 4

DEFF Research Database (Denmark)

Sækmose, Susanne Gjørup; Schlosser, Anders; Holst, René

2013-01-01

Microfibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation...... in systemic MFAP4 (sMFAP4) has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing...

Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

International Nuclear Information System (INIS)

Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

1988-01-01

Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer

Quantum Dot Nanotoxicity Investigations Using Human Lung Cells and TOXOR Electrochemical Enzyme Assay Methodology.

Science.gov (United States)

O'Hara, Tony; Seddon, Brian; O'Connor, Andrew; McClean, Siobhán; Singh, Baljit; Iwuoha, Emmanuel; Fuku, Xolile; Dempsey, Eithne

2017-01-27

Recent studies have suggested that certain nanomaterials can interfere with optically based cytotoxicity assays resulting in underestimations of nanomaterial toxicity. As a result there has been growing interest in the use of whole cell electrochemical biosensors for nanotoxicity applications. Herein we report application of an electrochemical cytotoxicity assay developed in house (TOXOR) in the evaluation of toxic effects of mercaptosuccinic acid capped cadmium telluride quantum dots (MSA capped CdTe QDs), toward mammalian cells. MSA capped CdTe QDs were synthesized, characterized, and their cytotoxicity toward A549 human lung epithelial cells investigated. The internalization of QDs within cells was scrutinized via confocal microscopy. The cytotoxicity assay is based on the measurement of changes in cellular enzyme acid phosphatase upon 24 h exposure to QDs. Acid phosphatase catalyzes dephosphorylation of 2-naphthyl phosphate to 2-naphthol (determined by chronocoulometry) and is indicative of metabolic activity in cells. The 24 h IC50 (concentration resulting in 50% reduction in acid phosphatase activity) value for MSA capped CdTe QDs was found to be 118 ± 49 μg/mL using the TOXOR assay and was in agreement with the MTT assay (157 ± 31 μg/mL). Potential uses of this electrochemical assay include the screening of nanomaterials, environmental toxins, in addition to applications in the pharmaceutical, food, and health sectors.

Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale

DEFF Research Database (Denmark)

Haven, Mai Østergaard; Lindedam, Jane; Jeppesen, Martin D.

2015-01-01

Recycling of enzymes in production of lignocellulosic bioethanol has been tried for more than 30 years. So far, the successes have been few and the experiments have been carried out at conditions far from those in an industrially feasible process. Here we have tested continuous enzyme recycling a...... broth also opens up the possibility of lowering the dry matter content in hydrolysis and fermentation while still maintaining high ethanol concentrations....... at demonstration scale using industrial process conditions (high dry matter content and low enzyme dosage) for a period of eight days. The experiment was performed at the Inbicon demonstration plant (Kalundborg, Denmark) capable of converting four tonnes of wheat straw per hour. 20% of the fermentation broth...... was recycled to the hydrolysis reactor while enzyme dosage was reduced by 5%. The results demonstrate that recycling enzymes by this method can reduce overall enzyme consumption and may also increase the ethanol concentrations in the fermentation broth. Our results further show that recycling fermentation...

A flow cytometric assay technology based on quantum dots-encoded beads

International Nuclear Information System (INIS)

Wang Haiqiao; Liu Tiancai; Cao Yuancheng; Huang Zhenli; Wang Jianhao; Li Xiuqing; Zhao Yuandi

2006-01-01

A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs' unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement

Palladium-Catalyzed Suzuki–Miyaura Cross-Coupling in Continuous Flow

Directory of Open Access Journals (Sweden)

Christophe Len

2017-05-01

Full Text Available Carbon–carbon cross-coupling reactions are among the most important processes in organic chemistry and Suzuki–Miyaura reactions are the most widely used protocols. For a decade, green chemistry and particularly catalysis and continuous flow, have shown immense potential in achieving the goals of “greener synthesis”. To date, it seems difficult to conceive the chemistry of the 21st century without the industrialization of continuous flow process in the area of pharmaceuticals, drugs, agrochemicals, polymers, etc. A large variety of palladium Suzuki–Miyaura cross-coupling reactions have been developed using a continuous flow sequence for preparing the desired biaryl derivatives. Our objective is to focus this review on the continuous flow Suzuki–Miyaura cross-coupling using homogeneous and heterogeneous catalysts.

Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

International Nuclear Information System (INIS)

Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

2006-01-01

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

Immunoglobulin G1 enzyme-linked Immunosorbent assay for diagnosis of Johne's disease in red deer (Cervus elaphus)

NARCIS (Netherlands)

Griffin, J.F.T.; Spittle, E.; Rodgers, C.R.; Liggett, S.; Cooper, M.; Bakker, D.; Bannantine, J.P.

2005-01-01

This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured

Quantification of patient specific assay interference in different formats of enzyme linked immunoassays for therapeutic monoclonal antibodies

NARCIS (Netherlands)

Grebenchtchikov, N.J.; Geurts-Moespot, A.; Heijmen, L.; Laarhoven, H.W.M. van; Herpen, C.M.L. van; Thijs, A.M.J.; Span, P.N.; Sweep, F.C.

2014-01-01

BackgroundThe use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are

Development of an enzyme-linked immunosorbent assay for the detection of dicamba.

Science.gov (United States)

Clegg, B S; Stephenson, G R; Hall, J C

2001-05-01

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in water. The CI-ELISA has a detection limit of 2.3 microg L(-1) and a linear working range of 10--10000 microg L(-1) with an IC(50) value of 195 microg L(-1). The dicamba polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with a dicamba metabolite, 5-hydroxydicamba, and structurally related chlorobenzoic acids. The assay was used to estimate quantitatively dicamba concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, a C(18) (reversed phase) column concentration step was devised prior to analysis, and the detection limits were increased by at least by 10-fold. After the sample preconcentration, the detection limit, IC(50), and linear working range were 0.23, 19.5, and 5-200 microg L(-1), respectively. The CI-ELISA estimations in water correlated well with those from gas chromatography-mass spectrometry (GC-MS) analysis (r(2) = 0.9991). This assay contributes to reducing laboratory costs associated with the conventional GC-MS residue analysis techniques for the quantitation of dicamba in water.

Vasoactive enzymes and blood flow responses to passive and active exercise in peripheral arterial disease

DEFF Research Database (Denmark)

Walker, Meegan A.; Høier, Birgitte; Walker, Philip J.

2016-01-01

Background: Peripheral arterial disease (PAD) is characterised by impaired leg blood flow, which contributes to claudication and reduced exercise capacity. This study investigated to what extent vasoactive enzymes might contribute to altered blood flow in PAD (Fontaine stage II). Methods: We...... compared femoral artery blood flow during reactive hyperaemia, leg-extension exercise and passive leg movement, and determined the level of vasoactive enzymes in skeletal muscle samples from the vastus lateralis in PAD (n = 10, 68.5 ± 6.5 years) and healthy controls (CON, n = 9, 62.1 ± 12.3 years). Leg...... than CON (1.04 ± 0.19 vs 0.50 ± 0.06 AU, P = 0.02), with no differences for other enzymes. Leg blood flow during exercise was correlated with prostacyclin synthase (P = 0.001). Conclusion: Elevated NADPH oxidase indicates that oxidative stress may be a primary cause of low nitric oxide availability...

Determination of PCBs in fish using enzyme-linked immunosorbent assay (ELISA)

Science.gov (United States)

Lasrado, J.A.; Santerre, C.R.; Zajicek, J.L.; Stahl, J.R.; Tillitt, D.E.; Deardorff, D.

2003-01-01

Polychlorinated biphenyls (PCBs) were determined in fish tissue using an enzyme-linked immunosorbent assay (ELISA). Standard curves for Aroclor 1248, 1254, and 1260 in catfish tissue were developed with ranges from 0.05 to 0.5 ppm and 0.5 to 5.0 ppm. Wild fish were initially analyzed using gas chromatography/electron-capture detection (GC/ECD) and those having residues within the standard curve ranges were analyzed with ELISA. Results obtained using ELISA and GC/ECD were not significantly different (p < 0.05) from 0.05 to 0.5 ppm. From 0.5 to 5.0 ppm, the standard curve for Aroclor 1254 was the best predictor of total PCB in wild fish samples.

Quantification of patient-specific assay interference in different formats of enzyme-linked immunoassays for therapeutic monoclonal antibodies

NARCIS (Netherlands)

Grebenchtchikov, Nicolai; Geurts-Moespot, Anneke J.; Heijmen, Linda; van Laarhoven, Hanneke W. M.; van Herpen, Carla M. L.; Thijs, Annemarie M. J.; Span, Paul N.; Sweep, Fred C. G. J.

2014-01-01

The use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are subject to a

Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

DEFF Research Database (Denmark)

Jensen, A T; Gaafar, A; Ismail, A

1996-01-01

An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...... samples from healthy Sudanese individuals living in an area endemic for malaria but free of leish-maniasis were negative in all the assays. Significantly higher levels of antibodies were found in the patients who had suffered from the disease for more than eight weeks than in patients with a shorter...

Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

Science.gov (United States)

Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...

The assembly and use of continuous flow systems for chemical synthesis.

Science.gov (United States)

Britton, Joshua; Jamison, Timothy F

2017-11-01

The adoption of and opportunities in continuous flow synthesis ('flow chemistry') have increased significantly over the past several years. Continuous flow systems provide improved reaction safety and accelerated reaction kinetics, and have synthesised several active pharmaceutical ingredients in automated reconfigurable systems. Although continuous flow platforms are commercially available, systems constructed 'in-lab' provide researchers with a flexible, versatile, and cost-effective alternative. Herein, we describe the assembly and use of a modular continuous flow apparatus from readily available and affordable parts in as little as 30 min. Once assembled, the synthesis of a sulfonamide by reacting 4-chlorobenzenesulfonyl chloride with dibenzylamine in a single reactor coil with an in-line quench is presented. This example reaction offers the opportunity to learn several important skills including reactor construction, charging of a back-pressure regulator, assembly of stainless-steel syringes, assembly of a continuous flow system with multiple junctions, and yield determination. From our extensive experience of single-step and multistep continuous flow synthesis, we also describe solutions to commonly encountered technical problems such as precipitation of solids ('clogging') and reactor failure. Following this protocol, a nonspecialist can assemble a continuous flow system from reactor coils, syringes, pumps, in-line liquid-liquid separators, drying columns, back-pressure regulators, static mixers, and packed-bed reactors.

Convective flow reversal in self-powered enzyme micropumps.

Science.gov (United States)

Ortiz-Rivera, Isamar; Shum, Henry; Agrawal, Arjun; Sen, Ayusman; Balazs, Anna C

2016-03-08

Surface-bound enzymes can act as pumps that drive large-scale fluid flows in the presence of their substrates or promoters. Thus, enzymatic catalysis can be harnessed for “on demand” pumping in nano- and microfluidic devices powered by an intrinsic energy source. The mechanisms controlling the pumping have not, however, been completely elucidated. Herein, we combine theory and experiments to demonstrate a previously unreported spatiotemporal variation in pumping behavior in urease-based pumps and uncover the mechanisms behind these dynamics. We developed a theoretical model for the transduction of chemical energy into mechanical fluid flow in these systems, capturing buoyancy effects due to the solution containing nonuniform concentrations of substrate and product. We find that the qualitative features of the flow depend on the ratios of diffusivities δ=D(P)/D(S) and expansion coefficients β=β(P)/β(S) of the reaction substrate (S) and product (P). If δ>1 and δ>β (or if δself-powered fluidic devices.

Process development of continuous glycerolysis in an immobilized enzyme-packed reactor for industrial monoacylglycerol production

DEFF Research Database (Denmark)

Damstrup, Marianne; Kiil, Søren; Jensen, Anker Degn

2007-01-01

Continuous and easily operated glycerolysis was studied in different lipase-packed columns to evaluate the most potential process set-ups for industrial monoacylglycerol (MAG) production. Practical design-related issues such as enzyme-filling degree, required reaction time, mass transfer investig......Continuous and easily operated glycerolysis was studied in different lipase-packed columns to evaluate the most potential process set-ups for industrial monoacylglycerol (MAG) production. Practical design-related issues such as enzyme-filling degree, required reaction time, mass transfer...

Probabilistic Power Flow Method Considering Continuous and Discrete Variables

Directory of Open Access Journals (Sweden)

Xuexia Zhang

2017-04-01

Full Text Available This paper proposes a probabilistic power flow (PPF method considering continuous and discrete variables (continuous and discrete power flow, CDPF for power systems. The proposed method—based on the cumulant method (CM and multiple deterministic power flow (MDPF calculations—can deal with continuous variables such as wind power generation (WPG and loads, and discrete variables such as fuel cell generation (FCG. In this paper, continuous variables follow a normal distribution (loads or a non-normal distribution (WPG, and discrete variables follow a binomial distribution (FCG. Through testing on IEEE 14-bus and IEEE 118-bus power systems, the proposed method (CDPF has better accuracy compared with the CM, and higher efficiency compared with the Monte Carlo simulation method (MCSM.

Evaluation of the radioimmunoassay, indirect enzyme linked immunosorbent assay, and dot blot assay for the identification of Xanthomonas campestris pv. phaseoli

Energy Technology Data Exchange (ETDEWEB)

Malin, E; Belden, E L; Roth, D

1985-09-01

A radioimmunoassay (RIA), an indirect competitive enzyme-linked immunosorbent assay (ELISA), and a dot-blot modification of the ELISA were evaluated for detection and identification of Xanthomonas campestris pv. phaseoli (X. c. pv. phaseoli). RIA and the dot blot tests were specific for X. c. pv. phaseoli; however, significant cross reactions occurred in the indirect competitive ELISA when using anti-X. c. pv. phaseoli antiserum against other closely related bacteria. The sensitivity level of all procedures for X. c. pv. phaseoli was approximately l0/sup 5/ colony forming unitsmL. All procedures were unsatisfactory in reliably detecting low levels of X. c. pv. phaseoli directly from extracts of bean seed. However when used in conjunction with ilution plating the dot blot assay and the RIA would be useful in specifically identifying X. c. pv. phaseoli. The relative merits of these tests for identification of X. c. pv. phaseoli are discussed.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

Science.gov (United States)

A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

Enzyme-Powered Pumps: From Fundamentals to Applications

Science.gov (United States)

Ortiz-Rivera, Isamar

, covering also the effect of the thermodynamics of the enzymatic reaction in the pumping behavior, and (3) the applicability of enzyme pumps as fluid flow-based inhibitor assays and as drug delivery devices. Our findings in each of these areas, gets us closer to our ultimate goal, where we aim to identify the optimal conditions needed for enzyme micropump operation, and construct a general model that could accurately predict enzyme micropump behavior for any enzyme-substrate combination. The information aforementioned has been divided in four chapters. Chapter 1 gives a quick glance into the development of enzyme-powered micropumps: from the systems and observed behaviors inspiring this work, to the first systems that were developed. The stability, duration, and extent of fluid pumping of enzyme pumps in general, are also discussed, along with the optimization of the enzyme-pump design. This chapter aims to provide a general idea of the motivation behind the concept of "enzyme-powered pumps", what are "enzyme-powered pumps", and which are the key features that characterize these systems. Chapter 2 is an extensive analysis of the mechanisms of actuation proposed for enzyme-powered micropumps. This chapter not only covers the first attempts to understand how enzyme pumps work, but also explores further the behavior of urease-powered pumps, which fluid flow patterns cannot be completely predicted only by considering thermal or solutal gradients. The findings of these studies could allow us to rationally control fluid flow for the directed delivery of payloads at designated locations. In Chapters 3 and 4, our focus was to highlight the potential application of enzyme-powered pumps for sensing and delivery. Chapter 3 explores the use of enzyme pumps as fluid flow-based inhibitor assays. At fixed concentrations of an enzyme and its substrate, the presence of an inhibitor can be detected by monitoring the decrease in fluid flow speed. Using this principle, sensors for toxic

An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

Science.gov (United States)

Joung, Hyou-Arm; Oh, Young Kyoung; Kim, Min-Gon

2014-03-15

Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3 ± 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum. © 2013 Elsevier B.V. All rights reserved.

Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E and other carboxypeptidase B-like enzymes

Energy Technology Data Exchange (ETDEWEB)

Fricker, L.D.; Devi, L. (Albert Einstein College of Medicine, Bronx, NY (USA))

1990-01-01

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg (125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity (125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10(-15) mol), compared to the fluorescent product (5 x 10(-11) mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described.

Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E and other carboxypeptidase B-like enzymes

International Nuclear Information System (INIS)

Fricker, L.D.; Devi, L.

1990-01-01

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg (125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity (125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10(-15) mol), compared to the fluorescent product (5 x 10(-11) mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described

Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

Science.gov (United States)

Kallmyer, Nathaniel E; Musielewicz, Joseph; Sutter, Joel; Reuel, Nigel F

2018-04-17

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

Directory of Open Access Journals (Sweden)

Jose E. VIDAL

2015-09-01

Full Text Available SUMMARYAIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex or enzyme-linked immunoassay (EIA has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered. CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcusspecies. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die.

An ultrafiltration assay for lysyl oxidase

International Nuclear Information System (INIS)

Shackleton, D.R.; Hulmes, D.J.

1990-01-01

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

The fluid mechanics of continuous flow electrophoresis

Science.gov (United States)

Saville, D. A.

1990-01-01

The overall objective is to establish theoretically and confirm experimentally the ultimate capabilities of continuous flow electrophoresis chambers operating in an environment essentially free of particle sedimentation and buoyancy. The efforts are devoted to: (1) studying the effects of particle concentration on sample conductivity and dielectric constant. The dielectric constant and conductivity were identified as playing crucial roles in the behavior of the sample and on the resolving power and throughput of continuous flow devices; and (2) improving the extant mathematical models to predict flow fields and particle trajectories in continuous flow electrophoresis. A dielectric spectrometer was designed and built to measure the complex dielectric constant of a colloidal dispersion as a function of frequency between 500 Hz and 200 kHz. The real part of the signal can be related to the sample's conductivity and the imaginary part to its dielectric constant. Measurements of the dielectric constants of several different dispersions disclosed that the dielectric constants of dilute systems of the sort encountered in particle electrophoresis are much larger than would be expected based on the extant theory. Experiments were carried out to show that, in many cases, this behavior is due to the presence of a filamentary structure of small hairs on the particle surface. A technique for producing electrokinetically ideal synthetic latex particles by heat treating was developed. Given the ubiquitous nature of hairy surfaces with both cells and synthetic particles, it was deemed necessary to develop a theory to explain their behavior. A theory for electrophoretic mobility of hairy particles was developed. Finally, the extant computer programs for predicting the structure of electro-osmotically driven flows were extended to encompass flow channels with variable wall mobilities.

Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products.

Science.gov (United States)

Zhang, Hongyan; Wang, Shuo

2009-10-31

The current status of enzyme-linked immunosorbent assays (ELISAs) for sulfonamides in edible animal products is reviewed. The attention was focused on the design and synthesis of haptens, conjugation to carrier protein, production of antibody, application of homologous and heterologous systems, as well as the molecular modeling of the haptens and sulfonamides. Researches have shown that sulfonamides seem to be particularly resistant to attempts to produce broad specificity antibodies. By summarizing the available research on sulfonamide ELISAs, it is hoped that it can be considered as a basis for further investigation aimed at developing the most efficient approaches for detection.

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

Science.gov (United States)

Bayramoglu, Gulay; Arica, M Yakup; Genc, Aysenur; Ozalp, V Cengiz; Ince, Ahmet; Bicak, Niyazi

2016-06-01

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.

Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

NARCIS (Netherlands)

Miners, J.S.; Verbeek, M.M.; Olde Rikkert, M.G.M.; Kehoe, P.G.; Love, S.

2008-01-01

Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and

[IFN-gamma enzyme-linked immunospot assay versus PPD tuberculin skin test in the diagnosis of tuberculous epididymitis].

Science.gov (United States)

Huang, Hao; Yang, Xi-Fei; Deng, Qun-Yi; Li, Bing; Liu, Guo-Hui; Zhang, Jie-Yun; Yang, Da-Fei

2012-06-01

To explore the potential application of IFN-gamma enzyme-linked immunospot (ELISPOT) assay in the diagnosis of tuberculous epididymitis (TE) by comparing ELISPOT assay with the traditional purified protein derivative (PPD) tuberculin skin test. We examined 13 TE patients using an in-house ELISPOT kit, another 11 TE patients by PPD skin testing, and 57 healthy male volunteers by parallel test with both the methods. Twelve (92.3%) of the 13 TE cases were positive on ELISPOT assay, and 10 (90.9%) of the 11 TE cases positive on PPD skin test, with no statistically significant differences between the two groups (P > 0.05). Among the 57 healthy male volunteers, 8 (14.0%) were positive on ELISPOT, and 28 (49.1%) positive on PPD test, the latter significantly higher than the former (P PPD test in the examination of tuberculous epididymitis. As for specificity, ELISPOT assay seems better than PPD test in differentiating tuberculous epididymitis patients from healthy males.

Immobilized enzyme studies in a microscale bioreactor.

Science.gov (United States)

Jones, Francis; Forrest, Scott; Palmer, Jim; Lu, Zonghuan; Elmore, John; Elmore, Bill B

2004-01-01

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.

An enzyme-linked immunosorbent assay for the quantification of serum platelet-bindable IgG.

Science.gov (United States)

Howe, S E; Lynch, D M; Lynch, J M

1984-01-01

An enzyme-linked immunosorbent assay (ELISA) using F(ab')2 peroxidase-labeled antihuman immunoglobulin and o-phenylenediamine dihydrochloride (OPD) as a substrate was developed to measure serum platelet bindable IgG (S-PBIgG). The assay was made quantitative by standardizing the number of normal "target" platelets bound to microtiter plate wells, and by incorporating quantitated IgG standards with each microtiter plate tested to prepare a standard calibration curve. By this method, S-PBIgG for normal individuals was 3.4 +/- 1.6 fg per platelet (mean +/- 1 SD; n = 40). Increased S-PBIgG levels were detected in 36 of 40 patients with clinical autoimmune thrombocytopenia (ATP), ranging from 7.0 to 85 fg per platelet. Normal S-PBIgG levels were found in 34 of 40 patients with nonimmune thrombocytopenia. This method showed a sensitivity of 90 percent, specificity of 85 percent, and in the sample population studied, a positive predictive value of 0.86 and a negative predictive value of 0.90. This assay is highly reproducible (coefficient of variation was 6.8%) and appears useful in the evaluation of patients with suspected immune-mediated thrombocytopenia.

Pin-count reduction for continuous flow microfluidic biochips

DEFF Research Database (Denmark)

Schneider, Alexander; Pop, Paul; Madsen, Jan

2017-01-01

Microfluidic biochips are replacing the conventional biochemical analyzers integrating the necessary functions on-chip. We are interested in flow-based biochips, where a continuous flow of liquid is manipulated using integrated microvalves, controlled from external pressure sources via off...

Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Zhao Hongwei; Xue Changguo; Nan Tiegui; Tan Guiyu; Li Zhaohu; Li, Qing X.; Zhang Qingchuan; Wang Baomin

2010-01-01

A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL -1 of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL -1 , respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

Enzyme-linked immunosorbent assay and radioimmunoassay of serum pepsinogen A

Energy Technology Data Exchange (ETDEWEB)

Pals, G; Meuwissen, S G.M.; Frants, R R; Kostense, P J; Eriksson, A W; Raesaenen, V

1987-02-01

The determination of serum pepsinogen A (=pepsinogen I) levels is of clinical importance in the study of duodenal ulcer, atrophic gastritis and gastric cancer. In the present study two different quantitative immunological techniques for serum pepsinogen A were compared: a radioimmunoassay (RIA) (Helsinki) and an enzyme-linked immunosorbent assay (ELISA) (Amsterdam). Serum samples of 177 subjects with various gastric diseases were tested in a double blind study. The correlation was excellent (r=0.954 in the range 0-760 ..mu..g/l and r=0.971 in the range 0-100 ..mu..g/l). The functional relationship between ELISA (x) and RIA (y), determined by weighted model II regression, was y=1.12x-0.54. Initially the use of goat anti-PGA in the ELISA resulted in falsely high values in about 10% of the individuals. This was caused by circulating antibodies cross-reacting with goat IgG. This artefact was eliminated by pre-incubation of all samples with non-immune goat serum.

Enzyme-linked immunosorbent assay and radioimmunoassay of serum pepsinogen A

International Nuclear Information System (INIS)

Pals, Gerard; Meuwissen, S.G.M.; Frants, R.R.; Kostense, P.J.; Eriksson, A.W.; Raesaenen, Vesa

1987-01-01

The determination of serum pepsinogen A (=pepsinogen I) levels is of clinical importance in the study of duodenal ulcer, atrophic gastritis and gastric cancer. In the present study two different quantitative immunological techniques for serum pepsinogen A were compared: a radioimmunoassay (RIA) (Helsinki) and an enzyme-linked immunosorbent assay (ELISA) (Amsterdam). Serum samples of 177 subjects with various gastric diseases were tested in a double blind study. The correlation was excellent (r=0.954 in the range 0-760 μg/l and r=0.971 in the range 0-100 μg/l). The functional relationship between ELISA (x) and RIA (y), determined by weighted model II regression, was y=1.12x-0.54. Initially the use of goat anti-PGA in the ELISA resulted in falsely high values in about 10% of the individuals. This was caused by circulating antibodies cross-reacting with goat IgG. This artefact was eliminated by pre-incubation of all samples with non-immune goat serum. (author)

Comparison among performances of a ligase chain reaction-based assay and two enzyme immunoassays in detecting Chlamydia trachomatis in urine specimens from men with nongonococcal urethritis.

Science.gov (United States)

Deguchi, T; Yasuda, M; Uno, M; Tada, K; Iwata, H; Komeda, H; Maeda, S; Latila, V; Saito, I; Kawada, Y

1996-01-01

We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis. PMID:8784574

A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

Directory of Open Access Journals (Sweden)

Gentry-Nielsen Martha J

2006-07-01

Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

The use of subcutaneous fat tissue for amyloid typing by enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Olsen, K E; Sletten, K; Westermark, Per

1999-01-01

for typing the most common systemic amyloidoses of AL, AA, and transthyretin types by enzyme-linked immunosorbent assay (ELISA), using abdominal wall subcutaneous fat biopsy specimens. The method was tested on 21 abdominal fat biopsy specimens that were sent to the laboratory. Of these, 15 contained amyloid......The amyloidoses are biochemically heterogeneous diseases with pathophysiologic deposits of various proteins. The clinical course, prognosis, and therapy are different for each type of amyloidosis and, therefore, a type-specific diagnosis is demanded as early as possible. We describe a method...

Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia

Directory of Open Access Journals (Sweden)

W. P. Lokapirnasari

2015-03-01

Full Text Available Aim: This study aims to produce and assay cellulolytic enzyme activity (endo-(1,4-β-D-glucanase, exo-(1,4-β-Dglucanase, and β-glucosidase, at optimum temperature and optimum pH of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. Materials and Methods: To produce enzyme from a single colony of E. cloacae WPL 214, 98 × 1010 CFU/ml of isolates was put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C in accordance with growth time and optimum temperature of E. cloacae WPL 214. Further on, culture was centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulose enzyme activity was withdrawn to assay endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase, and β-glucosidase. Results: Cellulase enzyme of E. cloacae WPL 214 isolates had endoglucanase activity of 0.09 U/ml, exoglucanase of 0.13 U/ml, and cellobiase of 0.10 U/ml at optimum temperature 35°C and optimum pH 5. Conclusion: E. cloacae WPL 214 isolated from bovine rumen fluid waste produced cellulose enzyme with activity as cellulolytic enzyme of endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase and β-glucosidase.

Inhibition of existing denitrification enzyme activity by chloramphenicol

Science.gov (United States)

Brooks, M.H.; Smith, R.L.; Macalady, D.L.

1992-01-01

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

Enzyme-linked immunosorbent Assay for detecting of antibody to canine distemper virus

Directory of Open Access Journals (Sweden)

Sudarisman

2006-03-01

Full Text Available Serum neutralisation test (SNT has been established for evaluating canine distemper vaccination, but until now SNT was rarely used due to the need for continuous tissue culture facilities and requires 3 days to perform. For detecting antibody to canine distemper virus, an enzyme-linked immunosorbent assay (ELISA is relatively simple and rapid seroassay. ELISA for canine immunoglobulin (Ig G antibodies to canine distemper virus (CDV was developed by using Onderstepoort strain of canine distemper virus as coating antigen. Rabbit anti canine IgG labelled with horse radish peroxidase was used as the conjugate, while phenylenediamine dihydrochloride (OPD was used as the substrate. The ELISA results were then compared with the results of the SNT, using the sera of 312 random-source dogs from West Java. The two test-results had a high degree of correlation. Very few discrepancies occurred and most of these were at the lower limits of each test. When the sera were tested at 1 : 100 dilutions, there was a 95.5% agreement between the ELISA and SNT. Their sensitivity and spesificity were 83.9 and 98.4%. Titrated SNT and ELISA also were performed on sera from 7 dogs whose lifetime medical histories were known. The antibodies were inclining up after two months of post vaccination, where the titre was not in zero/lower position at the day of vaccination. However, antibody zero or low position were found at 28 days post vaccination. All of the results indicated that ELISA can be used for evaluating antibody to canine distemper virus response, replacing the SNT.

Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Andresen, Lars Ole; Klausen, Joan; Barfod, Kristen

2002-01-01

and from herds declared free of infection with Ap. The Ap serotype 12 blocking ELISA showed a herd sensitivity of 0.77 (95% confidence interval, 0.62-0.88) and a herd specificity of 1.00 (0.95-1.00) with a cut-off value at 40% relative absorbance or 60% inhibition. The assay may be used advantageously......The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen...... in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotypc 12 infection...

Enhancing chemical synthesis using catalytic reactions under continuous flow conditions

OpenAIRE

Asadi, Mousa

2017-01-01

Many advantages have been demonstrated for continuous flow chemistry in comparison with batch chemistry; such as easy automation, high level of reproducibility, improved safety, and process reliability. Indeed, with continuous flow processes constant reaction parameters such as temperature, time, amount of reagents, catalyst, solvents, efficient mixing etc. can easily be assured. The research detailed in this PhD thesis takes advantages of flow chemistry applying it to the Fukuyama ...

Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

NARCIS (Netherlands)

Damen, Carola W. N.; de Groot, Els R.; Heij, Marianne; Boss, David S.; Schellens, Jan H. M.; Rosing, Hilde; Beijnen, Jos H.; Aarden, Lucien A.

2009-01-01

Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to

SERO-DIAGNOSIS OF TUBERCULOSIS WITH A60 ANTIGEN ENZYME-LINKED-IMMUNOSORBENT-ASSAY - FAILURE IN HIV-INFECTED INDIVIDUALS IN GHANA

NARCIS (Netherlands)

VANDERWERF, TS; DAS, PK; VANSOOLINGEN, D; YONG, S; VANDERMARK, TW

In order to assess the diagnostic usefulness of the A60 (ANDA Biologicals, Strassbourg, France) sero-diagnostic enzyme-linked immunosorbent assay (ELISA) kit for tuberculosis in Africa, sera of 53 pulmonary smear-positive tuberculosis (TB) patients, 30 apparently healthy control subjects and 6 AIDS

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

International Nuclear Information System (INIS)

Muir, D.; Varon, S.; Manthorpe, M.

1990-01-01

We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

Energy Technology Data Exchange (ETDEWEB)

Muir, D.; Varon, S.; Manthorpe, M. (Univ. of California, San Diego, La Jolla (USA))

1990-03-01

We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

Two-stage in vitro digestibility assay, a tool for formulating non-starch polysaccharide degrading enzyme combinations for commonly used feed ingredients of poultry rations

Directory of Open Access Journals (Sweden)

Y. Ramana Reddy

2013-05-01

Full Text Available Aim: An attempt was made to assess the effect of pure enzyme combinations with the objective of formulating customized enzyme mixtures based on sugar release when subjected to two-stage in vitro digestion assay. Materials and Methods: A two-stage in vitro digestibility assay was carried out for commonly used feed ingredients for poultry viz., maize, soy bean meal, sunflower cake, and de-oiled rice bran supplemented with three concentrations of xylanase (5000; 7500 and 10000 IU/kg, cellulase (50; 100 and 400 IU/kg and â-D-glucanase (100; 200 and 400 IU/kg were used to formulate various NSP enzymes combinations. In total 27 NSP enzyme combinations (3x3x3 were formulated and the sugar released due to NSP digestion was quantified by phenol sulphuric acid method. Results: The total sugar release was significantly (P<0.05 higher with supplementation of various enzymes combinations for maize, sunflower cake and de-oiled rice bran where as no significant (P<0.05 interaction of various NSP enzymes combinations was observed for soy bean meal. The NSP digestibility was highest in combination (xylanase-5000, cellulase-50 and â-D-glucanase-400 IU/kg, (xylanase-10000, cellulase-50 and â-D-glucanase-200 IU/kg and (xylanase-7500, cellulase- 100 and â-D-glucanase-100 IU/kg for maize, sunflower cake and de-oiled rice bran respectively. In case of sunflower cake, significant (P<0.01 three way interaction was observed among the xylanase, cellulose, and â-D-glucanase enzymes and the two-way interactions between the enzymes were also significant (P<0.01. Conclusion: It is concluded that 'n' number of non-starch Polysaccharide enzymes combinations can be screened for their efficiency to digest non-starch Polysaccharides present in various feed ingredients commonly used in poultry rations by employing two-stage in vitro digestibility assay as a tool. [Vet World 2013; 6(8.000: 525-529

Flow Injection Analysis

DEFF Research Database (Denmark)

Hansen, Elo Harald

1998-01-01

Learning objectives:* To provide an introduction to automated assays* To describe the basic principles of FIA * To demonstrate the capabilities of FIA in relation to batch assays and conventional continuous flow systems* To show that FIA allows one to augment existing analytical techniques* To sh...... how FIA offers novel analytical procedures which are not feasible by conventional means* To hightlight the potentials of FIA in selected practical assays...

Interaction of Thermus thermophilus ArsC enzyme and gold nanoparticles naked-eye assays speciation between As(III) and As(V)

International Nuclear Information System (INIS)

Politi, Jane; De Stefano, Luca; Spadavecchia, Jolanda; Casale, Sandra; Fiorentino, Gabriella; Antonucci, Immacolata

2015-01-01

The thermophilic bacterium Thermus thermophilus HB27 encodes chromosomal arsenate reductase (TtArsC), the enzyme responsible for resistance to the harmful effects of arsenic. We report on adsorption of TtArsC onto gold nanoparticles for naked-eye monitoring of biomolecular interaction between the enzyme and arsenic species. Synthesis of hybrid biological–metallic nanoparticles has been characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV–vis), dynamic light scattering (DLS) and phase modulated infrared reflection absorption (PM-IRRAS) spectroscopies. Molecular interactions have been monitored by UV–vis and Fourier transform-surface plasmon resonance (FT-SPR). Due to the nanoparticles’ aggregation on exposure to metal salts, pentavalent and trivalent arsenic solutions can be clearly distinguished by naked-eye assay, even at 85 μM concentration. Moreover, the assay shows partial selectivity against other heavy metals. (paper)

Micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

Energy Technology Data Exchange (ETDEWEB)

Layton, G T [Royal Infirmary, Manchester (UK)

1980-10-01

Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10/sup -3/ units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus.

Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

Science.gov (United States)

Wu, John T.Y.; Dreger, Sally; Chow, Eva Y.W.; Bowlby, Evelyn E.

2002-01-01

Abstract This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV−) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV− values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures. PMID:12418782

AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D)

Science.gov (United States)

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk pl...

Development of a Multianalyte Enzyme-Linked Immunosorbent Assay for Permethrin and Aroclors and Its Implementation for Analysis of Soil/Sediment and House Dust ExtractsExtracts

Science.gov (United States)

Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254, and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors ...

Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors

Energy Technology Data Exchange (ETDEWEB)

Mandenius, C F; Buelow, L; Danielsson, B; Mosbach, K

1985-02-01

Previously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injection of 50 ..mu..l samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucrose analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10-50 m degrees C for concentration of 0.05-0.30 M glucose or sucrose present in the original non-diluted samples. The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of sucrose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant. On-line control of the effluent concentration of glucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomputer. Present concentration of glucose in the effluent could be maintained over an extended period of time despite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance. 32 references.

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

Roth, G.J.; Machuga, E.T.

1982-01-01

Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

Activity assessment of microbial fibrinolytic enzymes.

Science.gov (United States)

Kotb, Essam

2013-08-01

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

Continuous microwave flow synthesis of mesoporous hydroxyapatite

International Nuclear Information System (INIS)

Akram, Muhammad; Alshemary, Ammar Z.; Goh, Yi-Fan; Wan Ibrahim, Wan Aini; Lintang, Hendrik O.; Hussain, Rafaqat

2015-01-01

We have successfully used continuous microwave flow synthesis (CMFS) technique for the template free synthesis of mesoporous hydroxyapatite. The continuous microwave flow reactor consisted of a modified 2.45 GHz household microwave, peristaltic pumps and a Teflon coil. This cost effective and efficient system was exploited to produce semi-crystalline phase pure nano-sized hydroxyapatite. Effect of microwave power, retention time and the concentration of reactants on the phase purity, degree of crystallinity and surface area of the final product was studied in detail. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were used to study the phase purity and composition of the product, while transmission electron microscopy (TEM) was used to study the effect of process parameters on the morphology of hydroxyapatite. The TEM analysis confirmed the formation of spherical particles at low microwave power; however the morphology of the particles changed to mesoporous needle and rod-like structure upon exposing the reaction mixture to higher microwave power and longer retention time inside the microwave. The in-vitro ion dissolution behavior of the as synthesized hydroxyapatite was studied by determining the amount of Ca 2+ ion released in SBF solution. - Highlights: • Continuous microwave flow synthesis method was used to prepare hydroxyapatite. • Increase in microwave power enhanced the degree of crystallinity. • TEM images confirmed the presence of mesopores on the surface of HA

Continuous microwave flow synthesis of mesoporous hydroxyapatite

Energy Technology Data Exchange (ETDEWEB)

Akram, Muhammad; Alshemary, Ammar Z.; Goh, Yi-Fan; Wan Ibrahim, Wan Aini [Department of Chemistry, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor (Malaysia); Lintang, Hendrik O. [Centre for Sustainable Nanomaterials (CSNano), Ibnu Sina Institute for Scientific and Industrial Research, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor (Malaysia); Hussain, Rafaqat, E-mail: rafaqat@kimia.fs.utm.my [Centre for Sustainable Nanomaterials (CSNano), Ibnu Sina Institute for Scientific and Industrial Research, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor (Malaysia)

2015-11-01

We have successfully used continuous microwave flow synthesis (CMFS) technique for the template free synthesis of mesoporous hydroxyapatite. The continuous microwave flow reactor consisted of a modified 2.45 GHz household microwave, peristaltic pumps and a Teflon coil. This cost effective and efficient system was exploited to produce semi-crystalline phase pure nano-sized hydroxyapatite. Effect of microwave power, retention time and the concentration of reactants on the phase purity, degree of crystallinity and surface area of the final product was studied in detail. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were used to study the phase purity and composition of the product, while transmission electron microscopy (TEM) was used to study the effect of process parameters on the morphology of hydroxyapatite. The TEM analysis confirmed the formation of spherical particles at low microwave power; however the morphology of the particles changed to mesoporous needle and rod-like structure upon exposing the reaction mixture to higher microwave power and longer retention time inside the microwave. The in-vitro ion dissolution behavior of the as synthesized hydroxyapatite was studied by determining the amount of Ca{sup 2+} ion released in SBF solution. - Highlights: • Continuous microwave flow synthesis method was used to prepare hydroxyapatite. • Increase in microwave power enhanced the degree of crystallinity. • TEM images confirmed the presence of mesopores on the surface of HA.

Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

Energy Technology Data Exchange (ETDEWEB)

Zhao Hongwei [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Xue Changguo [Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027 (China); Nan Tiegui; Tan Guiyu; Li Zhaohu [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Li, Qing X. [Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822 (United States); Zhang Qingchuan, E-mail: zhangqc@ustc.edu.cn [Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027 (China); Wang Baomin, E-mail: wbaomin@263.com [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China)

2010-08-31

A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL{sup -1} of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL{sup -1}, respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles

Science.gov (United States)

Guo, Longhua; Xu, Shaohua; Ma, Xiaoming; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

2016-09-01

Colorimetric enzyme-linked immunosorbent assay utilizing 3‧-3-5‧-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3‧-3-5‧-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

Science.gov (United States)

Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

2017-10-23

Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

Detection of antibodies to the extractable nuclear antigens by enzyme linked immunosorbent assay

International Nuclear Information System (INIS)

Aziz, Khalil A.; Fzizal, Abul A.

2005-01-01

Anti-extractable nuclear antigen (ENA) antibodies are a group of autoantibodies that are directed against various components of the cell nucleus. Antibodies to these antigens are closely associated with connective tissue disease. Early diagnosis of these diseases can prove very difficult and therefore clinicians rely on the use of anti-ENA antibody testing for the exclusion. Old methods of testing are time consuming and require great skills. For these reasons clinical immunology laboratories are switching to testing for anti-ENA antibodies by enzyme linked immunosorbent assay (ELISA). The latter assays are more sensitive and require little skills. In the present study we have investigated a number of different ELISA preparations. The study was conducted at Birmingham Heartlands Hospital during the period 2003. We tested a number of ENA-positive and negative samples using 3 different commercial ELISA preparations and compared the results with traditional CCIE-assay. The present study revealed that some ELISA preparations can be more sensitive than CCIE method. Laboratories still using later method should switch to ELISA. However it is important that laboratories evaluate a long range of different ELISA preparations before selecting the most optimal one. In addition it is recommended that laboratories then audit results in order to determine true significance of such results. Finally until the true significance of ELISA generated results is known, positive ENA-results should be interpreted in conjunction with the clinical picture and this would require close liaison in between the clinical immunology laboratory and clinicians

Reversible logic gates based on enzyme-biocatalyzed reactions and realized in flow cells: a modular approach.

Science.gov (United States)

Fratto, Brian E; Katz, Evgeny

2015-05-18

Reversible logic gates, such as the double Feynman gate, Toffoli gate and Peres gate, with 3-input/3-output channels are realized using reactions biocatalyzed with enzymes and performed in flow systems. The flow devices are constructed using a modular approach, where each flow cell is modified with one enzyme that biocatalyzes one chemical reaction. The multi-step processes mimicking the reversible logic gates are organized by combining the biocatalytic cells in different networks. This work emphasizes logical but not physical reversibility of the constructed systems. Their advantages and disadvantages are discussed and potential use in biosensing systems, rather than in computing devices, is suggested. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rat Liver Enzyme Release Depends on Blood Flow-Bearing Physical Forces Acting in Endothelium Glycocalyx rather than on Liver Damage

Directory of Open Access Journals (Sweden)

Julieta A. Díaz-Juárez

2017-01-01

Full Text Available We have found selective elevation of serum enzyme activities in rats subjected to partial hepatectomy (PH, apparently controlled by hemodynamic flow-bearing physical forces. Here, we assess the involvement of stretch-sensitive calcium channels and calcium mobilization in isolated livers, after chemical modifications of the endothelial glycocalyx and changing perfusion directionality. Inhibiting in vivo protein synthesis, we found that liver enzyme release is influenced by de novo synthesis of endothelial glycocalyx components, and released enzymes are confined into a liver “pool.” Moreover, liver enzyme release depended on extracellular calcium entry possibly mediated by stretch-sensitive calcium channels, and this endothelial-mediated mechanotransduction in liver enzyme release was also evidenced by modifying the glycocalyx carbohydrate components, directionality of perfusing flow rate, and the participation of nitric oxide (NO and malondialdehyde (MDA, leading to modifications in the intracellular distribution of these enzymes mainly as nuclear enrichment of “mitochondrial” enzymes. In conclusion, the flow-induced shear stress may provide fine-tuned control of released hepatic enzymes through mediation by the endothelium glycocalyx, which provides evidence of a biological role of the enzyme release rather to be merely a biomarker for evaluating hepatotoxicity and liver damage, actually positively influencing progression of liver regeneration in mammals.

Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

Science.gov (United States)

This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

Science.gov (United States)

Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

2016-01-01

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Akiyama, Mitoshi; Takaishi, Masatoshi; Murakami, Yoshie; Ueda, Ryuzo; Yamakido, Michio; Tsubokura, Tokuo.

1989-09-01

Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

Use of the Brucella IgM and IgG flow assays in the serodiagnosis of human brucellosis in an area endemic for brucellosis

NARCIS (Netherlands)

Irmak, Hasan; Buzgan, Turan; Evirgen, Omer; Akdeniz, Hayrettin; Demiroz, A. Pekcan; Abdoel, Theresia H.; Smits, Henk L.

2004-01-01

The clinical utility of two complementary tests for brucellosis, the Brucella IgM and IgG flow assays, was evaluated in a hospital in eastern Turkey. The results show that the flow assays are convenient diagnostic tests for use in endemic areas. A positive result in the flow assays was obtained in

Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA

Directory of Open Access Journals (Sweden)

Paula Ciaurriz

2017-01-01

Full Text Available The enzyme-linked immunosorbent assay (ELISA technique is based on the specific recognition ability of the molecular structure of an antigen (epitope by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs as a vehicle for secondary antibodies and peroxidase (HRP. The design of experiments technique (DOE and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof. As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.

A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

International Nuclear Information System (INIS)

Layton, G.T.

1980-01-01

Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10 -3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

Directory of Open Access Journals (Sweden)

Helene Andersson-Svahn

2011-11-01

Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins

International Nuclear Information System (INIS)

Huang Yong; Shi Ruina; Zhong Xuefei; Wang Dan; Zhao Meiping; Li Yuanzong

2007-01-01

The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins

Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

Science.gov (United States)

Reid, Michael S; Le, X Chris; Zhang, Hongquan

2018-04-27

Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a biomimetic enzyme-linked immunosorbent assay based on molecularly imprinted polymers on paper for the detection of carbaryl.

Science.gov (United States)

Zhang, Can; Cui, Hanyu; Han, Yufeng; Yu, Fangfang; Shi, Xiaoman

2018-02-01

A biomimetic enzyme-linked immunosorbent assay (BELISA) which was based on molecularly imprinted polymers on paper (MIPs-paper) with specific recognition was developed. As a detector, the surface of paper was modified with γ-MAPS by hydrolytic action and anchored the MIP layer on γ-MAPS modified-paper by copolymerization to construct the artificial antibody Through a series of experimentation and verification, we successful got the MIPs-paper and established BELISA for the detection of carbaryl. The development of MIPs-paper based on BELISA was applied to detect carbaryl in real samples and validated by an enzyme-linked immunosorbent assay (ELISA) based on anti-carbaryl biological antibody. The results of these two methods (BELISA and ELISA) were well correlated (R 2 =0.944). The established method of MIPs-paper BELISA exhibits the advantages of low cost, higher stability and being re-generable, which can be applied as a convenient tool for the fast and efficient detection of carbaryl. Copyright © 2017. Published by Elsevier Ltd.

Assay of ribulose bisphosphate carboxylase

International Nuclear Information System (INIS)

Pike, C.; Berry, J.

1987-01-01

Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, [ 14 C]-NaHCO 3 , and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30 0 . The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number

Natural Length Scales Shape Liquid Phase Continuity in Unsaturated Flows

Science.gov (United States)

Assouline, S.; Lehmann, P. G.; Or, D.

2015-12-01

Unsaturated flows supporting soil evaporation and internal drainage play an important role in various hydrologic and climatic processes manifested at a wide range of scales. We study inherent natural length scales that govern these flow processes and constrain the spatial range of their representation by continuum models. These inherent length scales reflect interactions between intrinsic porous medium properties that affect liquid phase continuity, and the interplay among forces that drive and resist unsaturated flow. We have defined an intrinsic length scale for hydraulic continuity based on pore size distribution that controls soil evaporation dynamics (i.e., stage 1 to stage 2 transition). This simple metric may be used to delineate upper bounds for regional evaporative losses or the depth of soil-atmosphere interactions (in the absence of plants). A similar length scale governs the dynamics of internal redistribution towards attainment of field capacity, again through its effect on hydraulic continuity in the draining porous medium. The study provides a framework for guiding numerical and mathematical models for capillary flows across different scales considering the necessary conditions for coexistence of stationarity (REV), hydraulic continuity and intrinsic capillary gradients.

An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes

Directory of Open Access Journals (Sweden)

Jing-Jing Wang

2014-08-01

Full Text Available An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam in the incubates were quantified using LC–MS/MS methods either by an internal standard (IS calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC50 values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition. Keywords: LC–MS/MS, Cytochrome P450, Cocktail-probe, Inhibition assessment, Drug screenning

Glypican-3 level assessed by the enzyme-linked immunosorbent assay is inferior to alpha-fetoprotein level for hepatocellular carcinoma diagnosis.

Science.gov (United States)

Jeon, Yejoo; Jang, Eun Sun; Choi, Yun Suk; Kim, Jin-Wook; Jeong, Sook-Hyang

2016-09-01

Glypican-3 (GPC3) protein is highly expressed in hepatocellular carcinoma (HCC) tissue. It has been suggested as a diagnostic biomarker, but its inconsistent performance means that it requires further assessment. We therefore investigated the diagnostic value of the plasma GPC3 level compared to the alpha-fetoprotein (AFP) level as a diagnostic biomarker of HCC. We enrolled 157 consecutive patients with newly diagnosed HCC and 156 patients with liver cirrhosis (LC) as the control group. GPC3 plasma levels were measured using two commercially available enzyme-linked immunosorbent assays (ELISAs, named as Assay 1 and 2), and AFP levels were measured using an enzyme-linked chemiluminescent immunoassay. The diagnostic accuracy was analyzed using the receiver operating characteristics (ROC) curve. Plasma GPC3 levels in HCC patients were very low (0-3.09 ng/mL) in Assay 1, while only 3 of the 157 patients (1.9%) showed detectable GPC3 levels in Assay 2. The median GPC3 level was not significantly elevated in the HCC group (0.80 ng/mL) compared with the LC group (0.60 ng/mL). The area under the ROC curve (AUC) for GPC3 was 0.559 in Assay 1. In contrast, the median AFP level was significantly higher in HCC (27.72 ng/mL) than in LC (4.74 ng/mL), with an AUC of 0.729. The plasma level of GPC3 is a poor diagnostic marker for HCC, being far inferior to AFP. The development of a consistent detection system for the blood level of GPC3 is warranted.

Evaluation of Two Commercial Enzyme-Linked Immunosorbent Assay Kits for the Detection of Human Circulating Metrnl.

Science.gov (United States)

Zheng, Si-Li; Li, Zhi-Yong; Zhang, Zheng; Wang, Dong-Sheng; Xu, Jian; Miao, Chao-Yu

2018-04-01

Metrnl is a newly discovered secreted protein with neurotrophic activity and metabolic effect, while in earlier studies its circulating level in human was not explored. We evaluated two commercial enzyme-linked immunosorbent assay kits (DY7867-05, R&D Systems and SK00478-02, Aviscera Bioscience) for the detection of human circulating Metrnl. The DY7867-05 kit showed superiority over the SK00478-02 kit since it generated better curve fitting degree, smaller variation among tests, higher inter-assay reproducibility and better specificity, and could effectively detect human Metrnl in six types of blood samples. Subsequent analysis was performed using the DY7867-05 kit. Sample storage conditions were investigated. No gender difference in circulating Metrnl levels was found, while people with newly diagnosed type 2 diabetes mellitus (T2DM) had significantly lower Metrnl levels compared to the healthy controls.

Controlled synthesis of poly(3-hexylthiophene in continuous flow

Directory of Open Access Journals (Sweden)

Helga Seyler

2013-07-01

Full Text Available There is an increasing demand for organic semiconducting materials with the emergence of organic electronic devices. In particular, large-area devices such as organic thin-film photovoltaics will require significant quantities of materials for device optimization, lifetime testing and commercialization. Sourcing large quantities of materials required for the optimization of large area devices is costly and often impossible to achieve. Continuous-flow synthesis enables straight-forward scale-up of materials compared to conventional batch reactions. In this study, poly(3-hexylthiophene, P3HT, was synthesized in a bench-top continuous-flow reactor. Precise control of the molecular weight was demonstrated for the first time in flow for conjugated polymers by accurate addition of catalyst to the monomer solution. The P3HT samples synthesized in flow showed comparable performance to commercial P3HT samples in bulk heterojunction solar cell devices.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

OpenAIRE

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. ...

A simple enzyme assay for dry matter digestibility and its value in studying food selection by generalist herbivores.

Science.gov (United States)

Choo, Gillian M; Waterman, Peter G; McKey, Doyle B; Gartlan, J Stephen

1981-05-01

The dry matter digestibility of 94 species of leaf was assayed by a simple method involving sequential treatment with pepsin and fungal cellulase enzymes. It was demonstrated that for foliage from rainforest trees of a wide range of dicotyledonous plant families the assay showed high positive correlation with estimates of dry matter digestibility obtained using rumenliquor from a fistulated steer. Both assays were found to reflect negative correlates of digestibility, notably fibre and condensed tannin, rather than the nutritional value of an item. The higher dry matter digestibility of immature leaves relative to mature leaves appeared to be accounted for by their lower fibre content. It is suggested that the pepsin/cellulase assay offers a cheap, quick, routine method of gaining information on the effects of some types of plant secondary compounds (digestibility reducers) on the 'food potential' of different kinds of foliage to herbivores. Its use in studies of herbivory in rainforest areas in relation to analyses for plant secondary compounds and food selection by herbivores is discussed.

Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues.

Science.gov (United States)

Zhang, Hongyan; Zhang, Yan; Wang, Shuo

2008-08-20

Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip-stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2-horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1-5 microg L(-1) or 1-10 microg L(-1) in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high-performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle (flow-through or dip-stick) or milk (only dip-stick).

Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); N. Juntti; B. Klingeborn; J. Groen (Jan); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1990-01-01

textabstractAn enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of

Improvement of an enzyme immunosorbent assay for detecting antibodies against Dioctophyma renale.

Science.gov (United States)

Pedrassani, Daniela; do Nascimento, Adjair Antonio; André, Marcos Rogério; Machado, Rosangela Zacarias

2015-09-15

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-Dioctophyma renale antibodies in the sera of dogs using, detection of parasite eggs in urine sediment as a reference test. ELISA uses a soluble antigenic preparation of esophagus of D. renale and the optimal dilutions of the antigen, serum and conjugate were determined by means of checker board titration, using positive (n=13) and negative (n=27) reference serum. The specificity and sensitivity of the ELISA were 93.8% and 92.3% respectively and the kappa index was good (0.76). These results suggest that ELISA described may prove to be an effective serological test for detecting dogs infected and exposed to this parasite mainly dogs that are not eliminating parasite eggs through their urine. Copyright © 2015 Elsevier B.V. All rights reserved.

Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

DEFF Research Database (Denmark)

Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

1995-01-01

We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay

OpenAIRE

Gulati, Baldev R.; Munir, Shirin; Patnayak, Devi P.; Goyal, Sagar M.; Kapur, Vivek

2001-01-01

The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detecti...

Quantification of urinary 5-hydroxyindoleacetic acid by in-house nitrosonaphthol reaction compared with nitrosonaphthol micro column chromatography and enzyme-linked immunosorbent assay

Directory of Open Access Journals (Sweden)

Joyce Matie Kinoshita da Silva

2014-06-01

Full Text Available The aim of this study was to compare the colorimetric "kit" and enzyme-linked immunosorbent assay (ELISA methods to quantify urinary 5-hydroxyindoleacetic acid through the Goldenberg's technique, exploring the potential of replacing it. 24-hour urine samples were tested by Goldenberg's assay and compared with kits. The agreement was almost perfect for the comparison of Goldenberg's assay with both colorimetric kit, and with ELISA kit, considering ≤ 7.5 mg/24h normal cutoff value. Therefore, both "kits" would be good alternatives to Goldenberg's technique due to practicality and agreement between values.

Continuous-Flow Biochips: Technology, Physical Design Methods and Testing

DEFF Research Database (Denmark)

Pop, Paul; Araci, Ismail Emre; Chakrabarty, Krishnendu

2015-01-01

This article is a tutorial on continuous-flow biochips where the basic building blocks are microchannels, and microvalves, and by combining them, more complex units such as mixers, switches, and multiplexers can be built. It also presents the state of the art in flow-based biochip technology...

Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten

2014-01-01

The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (m......Abs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal...... diseases....

Physical modeling of shoreline bioremediation: Continuous flow mesoscale basins

International Nuclear Information System (INIS)

Sveum, P.; Ramstad, S.; Faksness, L.G.; Bech, C.; Johansen, B.

1995-01-01

This paper describes the design and use of continuous flow basin beach models in the study of bioremediation processes, and gives some results from an experiment designed to study the effects of different strategies for adding fertilizers. The continuous flow experimental basin system simulates an open system with natural tidal variation, wave action, and continuous supply and exchange of seawater. Biodegradation and bioremediation processes can thus be tested close to natural conditions. Results obtained using the models show a significant enhancement of biodegradation of oil in a sediment treated with an organic nutrient source, increased nutrient level in the interstitial water, and sediment microbial activity. These physical models gives biologically significant results, and can be used to simulate biodegradation and bioremediation in natural systems

Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S)

DEFF Research Database (Denmark)

Leeming, Diana J; Nielsen, Mette J; Dai, Yueqin

2012-01-01

Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P type IV collagen...... expression in BDL rats (r = 0.49, P serum assay specific for P4NP 7S was highly related to liver fibrosis...

Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes.

Science.gov (United States)

Gard, A L; Warrington, A E; Pfeiffer, S E

1988-05-01

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.

Visualization periodic flows in a continuously stratified fluid.

Science.gov (United States)

Bardakov, R.; Vasiliev, A.

2012-04-01

To visualize the flow pattern of viscous continuously stratified fluid both experimental and computational methods were developed. Computational procedures were based on exact solutions of set of the fundamental equations. Solutions of the problems of flows producing by periodically oscillating disk (linear and torsion oscillations) were visualized with a high resolutions to distinguish small-scale the singular components on the background of strong internal waves. Numerical algorithm of visualization allows to represent both the scalar and vector fields, such as velocity, density, pressure, vorticity, stream function. The size of the source, buoyancy and oscillation frequency, kinematic viscosity of the medium effects were traced in 2D an 3D posing problems. Precision schlieren instrument was used to visualize the flow pattern produced by linear and torsion oscillations of strip and disk in a continuously stratified fluid. Uniform stratification was created by the continuous displacement method. The buoyancy period ranged from 7.5 to 14 s. In the experiments disks with diameters from 9 to 30 cm and a thickness of 1 mm to 10 mm were used. Different schlieren methods that are conventional vertical slit - Foucault knife, vertical slit - filament (Maksoutov's method) and horizontal slit - horizontal grating (natural "rainbow" schlieren method) help to produce supplementing flow patterns. Both internal wave beams and fine flow components were visualized in vicinity and far from the source. Intensity of high gradient envelopes increased proportionally the amplitude of the source. In domains of envelopes convergence isolated small scale vortices and extended mushroom like jets were formed. Experiments have shown that in the case of torsion oscillations pattern of currents is more complicated than in case of forced linear oscillations. Comparison with known theoretical model shows that nonlinear interactions between the regular and singular flow components must be taken

Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis

NARCIS (Netherlands)

Smits, Henk L.; Abdoel, Theresia H.; Solera, Javier; Clavijo, Encarnacion; Diaz, Ramon

2003-01-01

To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG

Electrohydrodynamics and other hydrodynamic phenomena in continuous-flow electrophoresis

International Nuclear Information System (INIS)

Saville, D.A.

1982-01-01

The process known as continuous flow electrophoresis employs an electric field to separate the constituents of particulate samples suspended in a liquid. Complications arise because the electric field generates temperature gradients due to Joule heating and derives an electrohydrodynamic crossflow. Several aspects of the flow are discussed including entrance effects, hydrodynamic stability and a flow restructuring due to the combined effects of buoyancy and the crossflow. 10 references

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

DEFF Research Database (Denmark)

Selman, L; Henriksen, M L; Brandt, J

2012-01-01

-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies....... The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined...... and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes....

Dot enzyme-linked immunosorbent assay (ELISA) for the detection of Toxocara infection using a rat model.

Science.gov (United States)

Paller, Vachel Gay V; Besana, Cyrelle M; Valdez, Isabel Kristine M

2017-12-01

Toxocariasis is a zoonotic disease usually caused by dog and cat roundworms, Toxocara canis and T. cati. Detection and diagnosis is difficult in paratenic and accidental hosts, including humans, as they cannot be detected through conventional methods such as fecal examination. Diagnosis therefore relies on immunological methods and molecular methods such as enzyme-linked immunosorbent assay (ELISA) and Western Blot, which are both time-consuming and requires sophisticated equipment. In the Philippines, only a few studies are available on Toxocara seroprevalence. Therefore, there is a need to adapt methods for serodiagnosis of Toxocara infection in humans for the Philippine setting. A dot enzyme linked immunosorbent assay (dot-ELISA) was standardized using T. canis excretory-secretory antigens. Test sera were collected from laboratory rats (Sprague-Dawley strain) experimentally infected with embryonated eggs of T. canis and Ascaris suum as well as rice field rats naturally infected with Taenia taeniaeformis and Nippostrongylus sp. Optimum conditions used were 20 µg/ml antigen concentration and 1:10 serum dilution. The sensitivity, specificity, positive, and negative predictive values were 90% (95% CI 55.5-99.7%), 100% (95% CI 69.2-100.0%), 100% (95% CI 66.4-100%), and 90.9% (95% CI 58.7-99.8%), respectively. Dot-ELISA has the potential to be developed as a cheaper, simpler, and more practical method for detection of anti- Toxocara antibodies on accidental hosts. This is a preliminary study conducted on experimental animals before optimization and standardization for human serum samples.

Development of a continuous-flow fluidic pump

International Nuclear Information System (INIS)

Robinson, S.M.

1985-08-01

A study was made of a fluidic pump which utilizes gas pistons, a venturi-like reverse-flow-diverter, and a planar Y-type flow junction to produce a continuous flow of liquid from a system containing no moving parts. The study included an evaluation of the system performance and of methods for controlling the stability of the fluidic system. A mathematical model of the system was developed for steady-state operation using accepted theories of fluid mechanics. Although more elaborate models are needed for detailed design and optimization of specific systems, the model determined some of the main factors controlling the system performance and will be used in the development of more accurate models. 49 refs., 39 figs., 9 tabs

Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay †

OpenAIRE

Wright, Sara F.; Morton, Joseph B.; Sworobuk, Janis E.

1987-01-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were no...

Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

Science.gov (United States)

Rodak, L; Babiuk, L A; Acres, S D

1982-07-01

The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

Synergistic Use of Gold Nanoparticles (AuNPs) and “Capillary Enzyme-Linked Immunosorbent Assay (ELISA)” for High Sensitivity and Fast Assays

Science.gov (United States)

Kim, Wan-Joong; Cho, Hyo Young; Jeong, Bongjin; Byun, Sangwon; Huh, JaeDoo; Kim, Young Jun

2017-01-01

Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min. PMID:29278402

Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps.

Science.gov (United States)

Harmsen, T; Jongerius, M C; van der Zwan, C W; Plantinga, A D; Kraaijeveld, C A; Berbers, G A

1992-01-01

A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA. PMID:1500523

Beyond organometallic flow chemistry : the principles behind the use of continuous-flow reactors for synthesis

NARCIS (Netherlands)

Noel, T.; Su, Y.; Hessel, V.; Noël, T.

2015-01-01

Flow chemistry is typically used to enable challenging reactions which are difficult to carry out in conventional batch equipment. Consequently, the use of continuous-flow reactors for applications in organometallic and organic chemistry has witnessed a spectacular increase in interest from the

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

Science.gov (United States)

Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

2018-06-04

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

Science.gov (United States)

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Development of an enzyme-linked immunosorbent assay-based method for measuring galactosyltransferase activity using a synthetic glycopolymer acceptor substrate.

Science.gov (United States)

Oubihi, M; Kitajima, K; Kobayashi, K; Adachi, T; Aoki, N; Matsuda, T

1998-03-15

A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring beta 1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a beta-linked N-acetylglucosamine (GlcNAc beta) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, beta-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by using Ricinus communis agglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAc beta), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.

Continuous microwave pasteurization of a vegetable smoothie improves its physical quality and hinders detrimental enzyme activity.

Science.gov (United States)

Arjmandi, Mitra; Otón, Mariano; Artés, Francisco; Artés-Hernández, Francisco; Gómez, Perla A; Aguayo, Encarna

2017-01-01

The effect of a pasteurization treatment at 90 ± 2 ℃ for 35 s provided by continuous microwave under different doses (low power/long time and high power/short time) or conventional pasteurization on the quality of orange-colored smoothies and their changes throughout 45 days of storage at 5 ℃ was investigated. A better color retention of the microwave pasteurization- treated smoothie using high power/short time than in conventionally processed sample was evidenced by the stability of the hue angle. The continuous microwave heating increased the viscosity of the smoothie more than the conventional pasteurization in comparison with non-treated samples. Lower residual enzyme activities from peroxidase, pectin methylesterase and polygalacturonase were obtained under microwave heating, specifically due to the use of higher power/shorter time. For this kind of smoothie, polygalacturonase was the more thermo-resistant enzyme and could be used as an indicator of pasteurization efficiency. The use of a continuous semi-industrial microwave using higher power and shorter time, such as 1600 W/206 s and 3600 W/93 s, resulted in better quality smoothies and greater enzyme reduction than conventional thermal treatment. © The Author(s) 2016.

Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Martin, Laurent; Chaabo, Ayman; Lasne, Françoise

2015-06-01

As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1-24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented. Copyright © 2014 John Wiley & Sons, Ltd.

Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Neiser, Susann; Koskenkorva, Taija S; Schwarz, Katrin; Wilhelm, Maria; Burckhardt, Susanna

2016-07-21

Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may

Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

Science.gov (United States)

Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

2008-09-15

Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

Science.gov (United States)

Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

2015-10-21

A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

The Generation of Diazo Compounds in Continuous-Flow.

Science.gov (United States)

Hock, Katharina J; Koenigs, Rene M

2018-03-25

Toxic, cancerogenic and explosive - these attributes are typically associated with diazo compounds. Nonetheless, diazo compounds are nowadays a highly demanded class of reagents for organic synthesis, yet the concerns with regards to safe and scalable transformations of these compounds are still exceptionally high. Lately, the research area of the continuous-flow synthesis of diazo compounds attracted significant interest and a whole variety of protocols for their "on-demand" preparation have been realized to date. This concept article focuses on the recent developments using continuous-flow technologies to access diazo compounds; thus minimizing risks and hazards when working with this particular class of compounds. In this article we discuss these concepts and highlight different pre-requisites to access and to perform downstream functionalization reaction. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections.

Science.gov (United States)

Galiana, Antonio; Coy, Javier; Gimeno, Adelina; Guzman, Noemi Marco; Rosales, Francisco; Merino, Esperanza; Royo, Gloria; Rodríguez, Juan Carlos

2017-01-01

Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients. In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays. Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines). SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively. This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship), in order for the results to be applied appropriately to the management of patients`infectious processes.

Multistep Continuous-Flow Synthesis in Medicinal Chemistry

DEFF Research Database (Denmark)

Petersen, Trine P; Mirsharghi, Sahar; Rummel, Pia Cwarzko

2013-01-01

A three-step continuous-flow synthesis system and its application to the assembly of a new series of chemokine receptor ligands directly from commercial building blocks is reported. No scavenger columns or solvent switches are necessary to recover the desired test compounds, which were obtained i...... studies in medicinal chemistry....

Diagnostic value of enzyme linked immuno-sorbent assay for cytomegalovirus disease.

Directory of Open Access Journals (Sweden)

Priya K

2002-07-01

Full Text Available BACKGROUND: Since interpretation of results of enzyme linked immuno-sorbent assay (ELISA for diagnosis of Cytomegalovirus (CMV infection in India is difficult, its diagnostic value required evaluation. AIMS: To evaluate the diagnostic value of ELISA against polymerase chain reaction (PCR in CMV disease. SETTINGS AND DESIGN: Results of ELISA test for CMV antibodies in CMV-DNA PCR positive and negative patients and normal healthy blood donors were analysed. METHODS AND MATERIAL: Anti-CMV antibodies were assayed by ELISA on the sera of 26 CMV PCR positive and 21 PCR negative patients and 35 normal healthy blood donors. STATISTICAL ANALYSIS: Chi square and Fischer exact test were used for statistical analysis. RESULTS: Anti-CMV antibodies (IgG or IgG and IgM were present in 20 (76.9% of 26 PCR positive and 13 (61.9% of 21 PCR negative patients. ELISA was negative in six (23.1% of 26 PCR positive patients. Of the 28 paediatric patients, ELISA was positive in 14 (73.7% of 19 PCR positive and three (33.3% of nine PCR negative patients showing a statistically significant difference (Chi square test, P value 0.038. Among the 19 patients having complications after organ transplant, ELISA showed anti-CMV antibodies in six (85.7% of seven PCR positive and 11 (91.7% of 12 PCR negative patients showing no significant difference. CMV-DNA was not detected in the buffy coat of 35 sero-positive blood donors. CONCLUSION: ELISA has no diagnostic value in the detection of CMV activation although it may help in the differential diagnosis of CMV infection in the paediatric age group.

Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens.

OpenAIRE

Ricke, S C; Schaefer, D M; Cook, M E; Kang, K H

1988-01-01

Cross-reactivity among four species of ruminal bacteria was examined by using egg yolk antibodies from immunized Leghorn laying hens and an enzyme-linked-immunosorbent assay. The effects of the four species on the hens were compared on various days postimmunization. Hens injected with the same bacterial species had similar apparent antibody levels over the entire postimmunization period, but only Bacteroides ruminicola B1(4) and Selenomonas ruminantium D antigens elicited early increases in a...

Thermomyces lanuginosus lipase-catalyzed synthesis of natural flavor esters in a continuous flow microreactor.

Science.gov (United States)

Gumel, Ahmad Mohammed; Annuar, M S M

2016-06-01

Enzymatic catalysis is considered to be among the most environmental friendly processes for the synthesis of fine chemicals. In this study, lipase from Thermomyces lanuginosus (Lecitase Ultra™) was used to catalyze the synthesis of flavor esters, i.e., methyl butanoate and methyl benzoate by esterification of the acids with methanol in a microfluidic system. Maximum reaction rates of 195 and 115 mM min -1 corresponding to catalytic efficiencies (k cat /K M ) of 0.30 and 0.24 min -1  mM -1 as well as yield conversion of 54 and 41 % were observed in methyl butanoate and methyl benzoate synthesis, respectively. Catalytic turnover (k cat ) was higher for methyl butanoate synthesis. Rate of synthesis and yield decreased with increasing flow rates. For both esters, increase in microfluidic flow rate resulted in increased advective transport over molecular diffusion and reaction rate, thus lower conversion. In microfluidic synthesis using T. lanuginosus lipase, the following reaction conditions were 40 °C, flow rate 0.1 mL min -1 , and 123 U g -1 enzyme loading found to be the optimum operating limits. The work demonstrated the application of enzyme(s) in a microreactor system for the synthesis of industrially important esters.

Effect of Flow Rate Controller on Liquid Steel Flow in Continuous Casting Mold using Numerical Modeling

Science.gov (United States)

Gursoy, Kadir Ali; Yavuz, Mehmet Metin

2014-11-01

In continuous casting operation of steel, the flow through tundish to the mold can be controlled by different flow rate control systems including stopper rod and slide-gate. Ladle changes in continuous casting machines result in liquid steel level changes in tundishes. During this transient event of production, the flow rate controller opening is increased to reduce the pressure drop across the opening which helps to keep the mass flow rate at the desired level for the reduced liquid steel level in tundish. In the present study, computational fluid dynamic (CFD) models are developed to investigate the effect of flow rate controller on mold flow structure, and particularly to understand the effect of flow controller opening on meniscus flow. First, a detailed validation of the CFD models is conducted using available experimental data and the performances of different turbulence models are compared. Then, the constant throughput casting operations for different flow rate controller openings are simulated to quantify the opening effect on meniscus region. The results indicate that the meniscus velocities are significantly affected by the flow rate controller and its opening level. The steady state operations, specified as constant throughput casting, do not provide the same mold flow if the controller opening is altered. Thus, for quality and castability purposes, adjusting the flow controller opening to obtain the fixed mold flow structure is proposed. Supported by Middle East Technical University (METU) BAP (Scientific Research Projects) Coordination.

Comparison of an enzyme linked immunosorbent assay (ELISA) and a radioallergosorbent test (RAST) for detection of IgE antibodies to Brugia malayi

NARCIS (Netherlands)

Wahyuni, Sitti; van Ree, Ronald; Mangali, Andarias; Supali, Taniawati; Yazdanbakhsh, Maria; Sartono, Erliyani

2003-01-01

The enzyme linked immunosorbent assay (ELISA) for specific IgE antibodies to Brugia malayi was compared with the radioallergosorbent test (RAST) for use in immunoepidemiological studies of lymphatic filariasis. Sera used were from individuals (aged 5-82 years) living in an area endemic for lymphatic

Evaluation of a commercial competitive enzyme-linked immunosorbent assay for detection of avian influenza virus subtype H5 antibodies in zoo birds

DEFF Research Database (Denmark)

Jensen, Trine Hammer; Andersen, Jannie Holmegaard; Hjulsager, Charlotte Kristiane

2017-01-01

The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study com...

A hydrogen fuel cell for rapid, enzyme-catalysed organic synthesis with continuous monitoring.

Science.gov (United States)

Wan, Lei; Megarity, Clare F; Siritanaratkul, Bhavin; Armstrong, Fraser A

2018-01-23

A one-pot fuel cell for specific, enzyme-catalysed organic synthesis, with continuous monitoring of rate and reaction progress, combines an electrode catalysing rapid, reversible and diffusion-controlled interconversion of NADP + and NADPH with a Pt electrode catalysing 2H + /H 2 interconversion. This Communication demonstrates its performance and characteristics using the reductive amination of 2-oxoglutarate as a test system.

Production of specific-structured lipids by enzymatic interesterification in a pilot continuous enzyme bed reactor

DEFF Research Database (Denmark)

Xu, Xuebing; Balchen, Steen; Høy, Carl-Erik

1998-01-01

Production of specific-structured lipids (interesterified lipids with a specific structure) by enzymatic interesterification was carried out in a continuous enzyme bed pilot scale reactor. Commercial immobilized lipase (Lipozyme IM) was used and investigations of acyl migration, pressure drop...

Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections.

Directory of Open Access Journals (Sweden)

Antonio Galiana

Full Text Available Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients.In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays.Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines. SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively.This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship, in order for the results to be applied appropriately to the management of patients`infectious processes.

Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

OpenAIRE

Alderete, J F

1984-01-01

An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses durin...

Superoxide Dismutase (SOD Enzyme Activity Assay in Fasciola spp. Para-sites and Liver Tissue Extract

Directory of Open Access Journals (Sweden)

M Assady

2011-09-01

Full Text Available Background: The purpose of this comparative study was to detect superoxide dismutase (SOD activities in Fasciola hepatica, F. gigantica parasites, infected and healthy liver tissues in order to determine of species effects and liver infection on SODs activity level.Methods: Fasciola spp. parasites and sheep liver tissues (healthy and infected liver tissues, 10 samples for each, were collected, homogenized and investigated for protein measurement, protein detection and SOD enzyme activity assay. Protein concentration was measured by Bradford method and SODs band protein was detected on SDS-PAGE. SODs activity was determined by iodonitrotetrazolium chloride, INT, and xanthine substrates. Independent samples t-test was conducted for analysis of SODs activities difference.Results: Protein concentration means were detected for F. hepatica 1.3 mg/ ml, F. gigantica 2.9 mg/ml, healthy liver tissue 5.5 mg/ml and infected liver tissue 1.6 mg/ml (with similar weight sample mass. Specific enzyme activities in the samples were obtained 0.58, 0.57, 0.51, 1.43 U/mg for F. hepatica, F. gigantica, healthy liver and infected liver respectively. Gel electrophoresis of Fasciola spp. and sheep liver tissue extracts revealed a band protein with MW of 60 kDa. The statistical analysis revealed significant difference between SOD activities of Fasciola species and also between SOD activity of liver tissues (P<.05.Conclusion: Fasciola species and liver infection are effective causes on SOD enzyme activity level.

Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

Science.gov (United States)

Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

2016-11-01

Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Separation of platelets from other blood cells in continuous-flow by dielectrophoresis field-flow-fractionation

OpenAIRE

Piacentini, Niccolò; Mernier, Guillaume; Tornay, Raphaël; Renaud, Philippe

2011-01-01

We present a microfluidic device capable of separating platelets from other blood cells in continuous flow using dielectrophoresis field-flow-fractionation. The use of hydrodynamic focusing in combination with the application of a dielectrophoretic force allows the separation of platelets from red blood cells due to their size difference. The theoretical cell trajectory has been calculated by numerical simulations of the electrical field and flow speed, and is in agreement with the experiment...

Applications of Continuous-Flow Photochemistry in Organic Synthesis, Material Science, and Water Treatment.

Science.gov (United States)

Cambié, Dario; Bottecchia, Cecilia; Straathof, Natan J W; Hessel, Volker; Noël, Timothy

2016-09-14

Continuous-flow photochemistry in microreactors receives a lot of attention from researchers in academia and industry as this technology provides reduced reaction times, higher selectivities, straightforward scalability, and the possibility to safely use hazardous intermediates and gaseous reactants. In this review, an up-to-date overview is given of photochemical transformations in continuous-flow reactors, including applications in organic synthesis, material science, and water treatment. In addition, the advantages of continuous-flow photochemistry are pointed out and a thorough comparison with batch processing is presented.

A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution

DEFF Research Database (Denmark)

Olsen, Johan Pelck; Kari, Jeppe; Borch, Kim

2017-01-01

of insoluble substrate. Perhaps for this reason, transient kinetics has rarely been reported for heterogeneous enzyme reactions. Here, we describe a quenched-flow system using peristaltic pumps and stirred substrate suspensions with a dead time below 100 ms. The general performance was verified by alkali...

Development and validation of an indirect Enzyme-linked Immunosorbent Assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants

NARCIS (Netherlands)

Heijden, van der H.M.J.F.; Bouwstra, R.J.; Mars, M.H.; Poel, van der W.H.M.; Wellenberg, G.J.; Maanen, van C.

2013-01-01

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood

A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies

International Nuclear Information System (INIS)

Marcusson-Staahl, Maritha; Cederbrant, Karin

2003-01-01

A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies. The classical 51 Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems. Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development. The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The final format of the assay includes 0.5 ml peripheral blood, 1.25x10 5 effector cells per sample, and collection of 500 target events by flow-cytometry. When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08%. The corresponding cytotoxic activity decreased from 68 to 8%. Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension. Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells. The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies. Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled

Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

International Nuclear Information System (INIS)

Dickinson, D.B.

1975-01-01

Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

Development of sensitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for monitoring bisphenol-A in canned foods and beverages.

Science.gov (United States)

Lu, Yang; Peterson, Joshua Richard; Gooding, John Justin; Lee, Nanju Alice

2012-06-01

Enzyme-linked immunosorbent assays (ELISAs) are investigated in this work for the detection of bisphenol-A (BPA), a plastic monomer and a critical contaminant in food and environment. A series of polyclonal antibodies generated in vivo using BPA-butyrate-protein conjugate and BPA-valerate-protein conjugate were evaluated on direct and indirect competitive assay formats with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate, and BPA-2-valerate). Two indirect ELISAs and one direct ELISA exhibiting high sensitivity and specificity for BPA were developed. The 50 % inhibition of antibody binding (IC(50)) values were 0.78 ± 0.01-1.20 ± 0.26 μg L(-1), and the limits of detection as measured by the IC(20) values were 0.10 ± 0.03-0.20 ± 0.04 μg L(-1). The assays were highly specific to BPA, only displaying low cross-reactivity (3-8 % for the indirect assays and 26 % for the direct assay) for 4-cumylphenol (4-CP), at pH 7.2. The degree of cross-reaction of 4-CP was influenced by the antibody/hapten conjugate combination, assay conditions, and the assay format. The assays were optimized for the analysis of BPA in canned vegetables, bottled water and carbonated drinks. The limits of quantification for these three evaluated sample types, based on the spike and recovery data, were 0.5, 2.5, and 100 μg L(-1), respectively.

Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

DEFF Research Database (Denmark)

Chow, E.Y.W.; Wu, J.T.Y.; Jauho, E.S.

2004-01-01

In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested.......9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between...

Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

Science.gov (United States)

Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

2017-09-01

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid.

Science.gov (United States)

Tabaei, Seyed R; Rabe, Michael; Zetterberg, Henrik; Zhdanov, Vladimir P; Höök, Fredrik

2013-09-25

Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

Measurement of Platelet-derived Microparticle Levels in the Chronic Phase of Cerebral Infarction Using an Enzyme-linked Immunosorbent Assay

OpenAIRE

Shirafuji, Toshihiko; Hamaguchi, Hirotoshi; Kanda, Fumio

2008-01-01

Assessment of platelet function is a critical component of the treatment andsecondary prevention of cerebral infarction, and measurement of platelet-derivedmicroparticle (PDMP) levels using flow cytometry may be a good indicator of plateletfunction. However, the flow cytometric analysis is not feasible in a variety of clinicalsituations. The goal of the present study was to measure PDMP levels using anenzyme-linked immunosorbent assay (ELISA) in chronic cerebral infarction patientsand to dete...

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

Science.gov (United States)

Rasmussen, M; Dahl, M; Buus, S; Djurisic, S; Ohlsson, J; Hviid, T V F

2014-08-01

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use. © 2014 John Wiley & Sons A/S. Published by John Wiley

A three-parameter langmuir-type model for fitting standard curves of sandwich enzyme immunoassays with special attention to the α-fetoprotein assay

NARCIS (Netherlands)

Kortlandt, W.; Endeman, H.J.; Hoeke, J.O.O.

In a simplified approach to the reaction kinetics of enzyme-linked immunoassays, a Langmuir-type equation y = [ax/(b + x)] + c was derived. This model proved to be superior to logit-log and semilog models in the curve-fitting of standard curves. An assay for α-fetoprotein developed in our laboratory

Extracellular enzyme activity in a willow sewage treatment system.

Science.gov (United States)

Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

2012-12-01

This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

Onderzoek naar toepasbaarheid van Enzyme-Linked Immunosorbent Assay (ELISA) voor het bepalen van aflatoxinen in voedings- en voedermiddelen 1e interimrapport: Orientatie en taakstelling

NARCIS (Netherlands)

Egmond; H.P. van; Paulsch; W.E.; Sizoo; E.A.

1987-01-01

Immunochemische methoden van onderzoek voor het bepalen van mycotoxinen komen langzamerhand in de belangstelling te staan, vooral Enzyme-Linked Immunosorbent Assay (ELISA). Om een beeld te krijgen van de praktische en wetenschappelijke karakteristieken van ELISA's in het

Initial in vitro testing of a paediatric continuous-flow total artificial heart.

Science.gov (United States)

Fukamachi, Kiyotaka; Karimov, Jamshid H; Horvath, David J; Sunagawa, Gengo; Byram, Nicole A; Kuban, Barry D; Moazami, Nader

2018-06-01

Mechanical circulatory support has become standard therapy for adult patients with end-stage heart failure; however, in paediatric patients with congenital heart disease, the options for chronic mechanical circulatory support are limited to paracorporeal devices or off-label use of devices intended for implantation in adults. Congenital heart disease and cardiomyopathy often involve both the left and right ventricles; in such cases, heart transplantation, a biventricular assist device or a total artificial heart is needed to adequately sustain both pulmonary and systemic circulations. We aimed to evaluate the in vitro performance of the initial prototype of our paediatric continuous-flow total artificial heart. The paediatric continuous-flow total artificial heart pump was downsized from the adult continuous-flow total artificial heart configuration by a scale factor of 0.70 (1/3 of total volume) to enable implantation in infants. System performance of this prototype was evaluated using the continuous-flow total artificial heart mock loop set to mimic paediatric circulation. We generated maps of pump performance and atrial pressure differences over a wide range of systemic vascular resistance/pulmonary vascular resistance and pump speeds. Performance data indicated left pump flow range of 0.4-4.7 l/min at 100 mmHg delta pressure. The left/right atrial pressure difference was maintained within ±5 mmHg with systemic vascular resistance/pulmonary vascular resistance ratios between 1.4 and 35, with/without pump speed modulation, verifying expected passive self-regulation of atrial pressure balance. The paediatric continuous-flow total artificial heart prototype met design requirements for self-regulation and performance; in vivo pump performance studies are ongoing.

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

DEFF Research Database (Denmark)

Jensen, A T; Gasim, S; Moller, T

1999-01-01

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...... for malaria but free of leishmaniasis was negative in both assays....

Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures

International Nuclear Information System (INIS)

Wohlleben, Wendel; Kolle, Susanne N; Hasenkamp, Laura-Carolin; Boeser, Alexander; Vogel, Sandra; Vacano, Bernhard von; Ravenzwaay, Ben van; Landsiedel, Robert

2011-01-01

We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

Practical steady-state enzyme kinetics.

Science.gov (United States)

Lorsch, Jon R

2014-01-01

Enzymes are key components of most biological processes. Characterization of enzymes is therefore frequently required during the study of biological systems. Steady-state kinetics provides a simple and rapid means of assessing the substrate specificity of an enzyme. When combined with site-directed mutagenesis (see Site-Directed Mutagenesis), it can be used to probe the roles of particular amino acids in the enzyme in substrate recognition and catalysis. Effects of interaction partners and posttranslational modifications can also be assessed using steady-state kinetics. This overview explains the general principles of steady-state enzyme kinetics experiments in a practical, rather than theoretical, way. Any biochemistry textbook will have a section on the theory of Michaelis-Menten kinetics, including derivations of the relevant equations. No specific enzymatic assay is described here, although a method for monitoring product formation or substrate consumption over time (an assay) is required to perform the experiments described. © 2014 Elsevier Inc. All rights reserved.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Science.gov (United States)

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Product differentiation during continuous-flow thermal gradient PCR.

Science.gov (United States)

Crews, Niel; Wittwer, Carl; Palais, Robert; Gale, Bruce

2008-06-01

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

Validation of an improved enzyme-linked immunosorbent assay for the diagnosis of trypanosomal antibodies in Ghanaian cattle

International Nuclear Information System (INIS)

Doku, C.K.; Seidu, I.B.M.

2000-01-01

The validation of an enzyme-linked immunosorbent assay (Ab-ELISA) for the detection of antibodies to pathogenic trypanosomes in cattle is described. Two hundred known negative sera obtained from the tsetse-free zone of Dori (Burkina Faso) were analyzed using microtitre plates pre-coated with crude antigen lysates of Trypanosoma congolense and T. vivax. A pre-test optimization was carried out and a percent positivity (PP) of 50% was chosen (specificity: >82%) for assaying field sera. A total of 440 serum samples collected from cattle in areas of known and unknown disease prevalence were assayed. For all animals the packed red cell volume (PCV) was determined and the buffy coat technique (BCT) and blood smears were examined to detect trypanosomes at the species level. A comparison of the BCT and Ab-ELISA results showed there was a much higher prevalence of antibodies to both species than the parasite prevalence as shown by the BCT (10 fold). The rate of agreement between BCT-positive and Ab-ELISA-positive samples for both species was low (<10%). No conclusion could be drawn from this finding because of the low number of known BCT positive cases that were identified. There was a better, albeit highly variable, agreement between BCT-negative and Ab-ELISA-negative samples (30-70%). Proposals for further improvement of the Ab-ELISA and prospects for the use of the assay in the monitoring of trypanosomosis control in Ghana are discussed. (author)

A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

Science.gov (United States)

Li, Ye; Cassone, Vincent M

2015-09-01

A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone. Copyright © 2015 Elsevier B.V. All rights reserved.

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

Science.gov (United States)

Alcorn, S.W.; Pascho, R.J.

2000-01-01

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

Design and testing of a unique randomized gravity, continuous flow bioreactor

Science.gov (United States)

Lassiter, Carroll B.

1993-01-01

A rotating, null gravity simulator, or Couette bioreactor was successfully used for the culture of mammalian cells in a simulated microgravity environment. Two limited studies using Lipomyces starkeyi and Streptomyces clavuligerus were also conducted under conditions of simulated weightlessness. Although these studies with microorganisms showed promising preliminary results, oxygen limitations presented significant limitations in studying the biochemical and cultural characteristics of these cell types. Microbial cell systems such as bacteria and yeast promise significant potential as investigative models to study the effects of microgravity on membrane transport, as well as substrate induction of inactive enzyme systems. Additionally, the smaller size of the microorganisms should further reduce the gravity induced oscillatory particle motion and thereby improve the microgravity simulation on earth. Focus is on the unique conceptual design, and subsequent development of a rotating bioreactor that is compatible with the culture and investigation of microgravity effects on microbial systems. The new reactor design will allow testing of highly aerobic cell types under simulated microgravity conditions. The described reactor affords a mechanism for investigating the long term effects of reduced gravity on cellular respiration, membrane transfer, ion exchange, and substrate conversions. It offers the capability of dynamically altering nutrients, oxygenation, pH, carbon dioxide, and substrate concentration without disturbing the microgravity simulation, or Couette flow, of the reactor. All progeny of the original cell inoculum may be acclimated to the simulated microgravity in the absence of a substrate or nutrient. The reactor has the promise of allowing scientists to probe the long term effects of weightlessness on cell interactions in plants, bacteria, yeast, and fungi. The reactor is designed to have a flow field growth chamber with uniform shear stress, yet transfer

Quantifying sublethal effects of glyphosate and Roundup® to Daphnia magna using a fluorescence based enzyme activity assay and video tracking

DEFF Research Database (Denmark)

Roslev, Peter; R. Hansen, Lone; Ørsted, Michael

Glyphosate (N-(phosphonomethyl)glycine) is the active ingredient in a range of popular broad-spectrum, non-selective herbicide formulations. The toxicity of this herbicide to non-target aquatic organisms such as Daphnia magna is often evaluated using conventional toxicity assays that focus...... on endpoints such as immobility and mortality. In this study, we investigated sublethal effects of glyphosate and Roundup® to D. magna using video tracking for quantifying behavioral changes, and a novel fluorescence based assay for measuring in vivo hydrolytic enzyme activity (FLEA assay). Roundup® exposure...... resulted in concentration-dependent inhibition of alkaline phosphatase activity in D. magna. The inhibition of alkaline phosphatase by Roundup® was temperature-dependent with lowest inhibition at 14 °C and greater inhibition at 20 and 26 °C. Exposure of D. magna to sublethal concentrations of glyphosate...

Continuous vs. pulsating flow boiling. Part 2: Statistical comparison using response surface methodology

DEFF Research Database (Denmark)

Kærn, Martin Ryhl; Elmegaard, Brian; Meyer, Knud Erik

2016-01-01

Response surface methodology is used to investigate an active method for flow boiling heat transfer enhancement by means of fluid flow pulsation. The flow pulsations are introduced by a flow modulating expansion device and compared with the baseline continuous flow provided by a stepper...

Influenza A plasma and serum virus antibody detection comparison in dogs using blocking enzyme-linked immunosorbent assay

Directory of Open Access Journals (Sweden)

H. T. Lin

2015-05-01

Full Text Available Background and Aim: The influenza A virus (IAV is an important zoonotic pathogen with infections also reported in dogs. IAV infections can be detected through the presence of antibodies using the enzyme-linked immunosorbent assay (ELISA. Serum is the only standard sample source; however, there is no information on the availability of other sample sources for IAV antibody detection in dogs. Compared with serum, plasma is more widely employed in most animal hospitals. The object of this study is to investigate whether plasma collected in ethylenediaminetetraacetic acid (EDTA tubes (EDTA plasma or heparin tubes (heparin plasma could be used in the ELISA protocol instead of serum for IAV antibody detection in dogs. Materials and Methods: Totally, 82 matched EDTA plasma and serum sample pairs and 79 matched heparin plasma and serum sample pairs were employed using blocking enzyme-linked immunosorbent assay (bELISA. The agreement and correlation between the plasma (EDTA or heparin plasma and serum were assessed using the agreement index kappa (kD calculation and Pearson correlation coefficient, respectively. Results: The agreement index kD of EDTA plasma and serum was 1.0, and that of heparin plasma and serum was 0.85. The Pearson correlation coefficient of EDTA plasma and serum was 0.87 (p<0.01, and that of heparin plasma and serum was 0.82 (p<0.01. Conclusion: The results proved that plasma, especially EDTA plasma, could be substituted for serum in the bELISA test. This might greatly expand the clinical applicability of IAV antibody detection in dogs.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

Directory of Open Access Journals (Sweden)

Kathleen Ingenhoven

2017-07-01

Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

Science.gov (United States)

Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

1998-12-01

Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

Hybrid Continuous-Flow Total Artificial Heart.

Science.gov (United States)

Fox, Carson; Chopski, Steven; Murad, Nohra; Allaire, Paul; Mentzer, Robert; Rossano, Joseph; Arabia, Francisco; Throckmorton, Amy

2018-05-01

Clinical studies using total artificial hearts (TAHs) have demonstrated that pediatric and adult patients derive quality-of-life benefits from this form of therapy. Two clinically-approved TAHs and other pumps under development, however, have design challenges and limitations, including thromboembolic events, neurologic impairment, infection risk due to large size and percutaneous drivelines, and lack of ambulation, to name a few. To address these limitations, we are developing a hybrid-design, continuous-flow, implantable or extracorporeal, magnetically-levitated TAH for pediatric and adult patients with heart failure. This TAH has only two moving parts: an axial impeller for the pulmonary circulation and a centrifugal impeller for the systemic circulation. This device will utilize the latest generation of magnetic bearing technology. Initial geometries were established using pump design equations, and computational modeling provided insight into pump performance. The designs were the basis for prototype manufacturing and hydraulic testing. The study results demonstrate that the TAH is capable of delivering target blood flow rates of 1-6.5 L/min with pressure rises of 1-92 mm Hg for the pulmonary circulation and 24-150 mm Hg for the systemic circulation at 1500-10 000 rpm. This initial design of the TAH was successful and serves as the foundation to continue its development as a novel, more compact, nonthrombogenic, and effective therapeutic alternative for infants, children, adolescents, and adults with heart failure. © 2018 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

DEFF Research Database (Denmark)

Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders

2015-01-01

Background: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradica...

Low sensitivity of type VII collagen enzyme-linked immunosorbent assay in epidermolysis bullosa acquisita : serration pattern analysis on skin biopsy is required for diagnosis

NARCIS (Netherlands)

Terra, J. B.; Jonkman, M. F.; Diercks, G. F. H.; Pas, H. H.

BackgroundThe type VII collagen (coll VII) enzyme-linked immunosorbent assay (ELISA) has been reported to have high sensitivity (>93%) and specificity (>96%) for diagnosing epidermolysis bullosa acquisita (EBA) in patients who are seropositive on indirect immunofluorescence on salt-split skin (SSS).

Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor.

Science.gov (United States)

Ringborg, Rolf H; Toftgaard Pedersen, Asbjørn; Woodley, John M

2017-09-08

Enzyme-mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube-in-tube reactor, operated in the low-dispersed flow regime to generate time-series data, with minimal material consumption. Experimental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high K MO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle.

Diels–Alder reactions of myrcene using intensified continuous-flow reactors

Directory of Open Access Journals (Sweden)

Christian H. Hornung

2017-01-01

Full Text Available This work describes the Diels–Alder reaction of the naturally occurring substituted butadiene, myrcene, with a range of different naturally occurring and synthetic dienophiles. The synthesis of the Diels–Alder adduct from myrcene and acrylic acid, containing surfactant properties, was scaled-up in a plate-type continuous-flow reactor with a volume of 105 mL to a throughput of 2.79 kg of the final product per day. This continuous-flow approach provides a facile alternative scale-up route to conventional batch processing, and it helps to intensify the synthesis protocol by applying higher reaction temperatures and shorter reaction times.

Harnessing Thin-Film Continuous-Flow Assembly Lines.

Science.gov (United States)

Britton, Joshua; Castle, Jared W; Weiss, Gregory A; Raston, Colin L

2016-07-25

Inspired by nature's ability to construct complex molecules through sequential synthetic transformations, an assembly line synthesis of α-aminophosphonates has been developed. In this approach, simple starting materials are continuously fed through a thin-film reactor where the intermediates accrue molecular complexity as they progress through the flow system. Flow chemistry allows rapid multistep transformations to occur via reaction compartmentalization, an approach not amenable to using conventional flasks. Thin film processing can also access facile in situ solvent exchange to drive reaction efficiency, and through this method, α-aminophosphonate synthesis requires only 443 s residence time to produce 3.22 g h(-1) . Assembly-line synthesis allows unprecedented reaction flexibility and processing efficiency. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzyme-linked immunosorbent assays for Z-DNA.

Science.gov (United States)

Thomas, M J; Strobl, J S

1988-10-01

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e.l.i.s.a. was conducted in 48-well culture dishes at 37 degrees C using a rabbit polyclonal antiserum developed against Br-poly(dG-dC).poly(dG-dC), an alkaline phosphatase-conjugated second antibody, and p-nitrophenol as the substrate. Under conditions where antibody concentrations were not limiting, alkaline phosphatase activity was linear for 2 h. Dot blot e.l.i.s.a. conditions are described which allow quantification of Z-DNA [Br-poly(dG-dC).poly(dG-dC)] within the range 5-250 ng. Dot blot and transblot horseradish peroxidase e.l.i.s.a. are described that detect Z-DNA within supercoiled plasmid DNAs immobilized on diazophenylthioether (DPT) paper. In the transblot e.l.i.s.a., plasmid pUC8 derivatives containing 16, 24, or 32 residues of Z-DNA were electrophoresed in agarose gels and electrophoretically transferred to DPT paper. Z-DNA-antibody complexes were detected by the horseradish peroxidase-catalysed conversion of 4-chloro-1-naphthol to a coloured product that was covalently bound to the DPT paper. Z-DNA antibody reactivity was specific for supercoiled Z-DNA containing plasmids after removal of the antibodies cross-reactive with B-DNA by absorption onto native DNA-cellulose. The transblot e.l.i.s.a. was sensitive enough to detect 16 base pairs of alternating G-C residues in 100 ng of pUC8 DNA.

Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

Science.gov (United States)

Cortina, María E; Novak, Analía; Melli, Luciano J; Elena, Sebastián; Corbera, Natalia; Romero, Juan E; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

2017-09-01

Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans. Copyright © 2017 Elsevier B.V. All rights reserved.

The synthesis of active pharmaceutical ingredients (APIs using continuous flow chemistry

Directory of Open Access Journals (Sweden)

Marcus Baumann

2015-07-01

Full Text Available The implementation of continuous flow processing as a key enabling technology has transformed the way we conduct chemistry and has expanded our synthetic capabilities. As a result many new preparative routes have been designed towards commercially relevant drug compounds achieving more efficient and reproducible manufacture. This review article aims to illustrate the holistic systems approach and diverse applications of flow chemistry to the preparation of pharmaceutically active molecules, demonstrating the value of this strategy towards every aspect ranging from synthesis, in-line analysis and purification to final formulation and tableting. Although this review will primarily concentrate on large scale continuous processing, additional selected syntheses using micro or meso-scaled flow reactors will be exemplified for key transformations and process control. It is hoped that the reader will gain an appreciation of the innovative technology and transformational nature that flow chemistry can leverage to an overall process.

The synthesis of active pharmaceutical ingredients (APIs) using continuous flow chemistry.

Science.gov (United States)

Baumann, Marcus; Baxendale, Ian R

2015-01-01

The implementation of continuous flow processing as a key enabling technology has transformed the way we conduct chemistry and has expanded our synthetic capabilities. As a result many new preparative routes have been designed towards commercially relevant drug compounds achieving more efficient and reproducible manufacture. This review article aims to illustrate the holistic systems approach and diverse applications of flow chemistry to the preparation of pharmaceutically active molecules, demonstrating the value of this strategy towards every aspect ranging from synthesis, in-line analysis and purification to final formulation and tableting. Although this review will primarily concentrate on large scale continuous processing, additional selected syntheses using micro or meso-scaled flow reactors will be exemplified for key transformations and process control. It is hoped that the reader will gain an appreciation of the innovative technology and transformational nature that flow chemistry can leverage to an overall process.

Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

Science.gov (United States)

Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

2003-01-01

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

Rapid continuous flow synthesis of high-quality silver nanocubes and nanospheres

KAUST Repository

Mehenni, Hakim

2013-01-01

We report a biphasic-liquid segmented continuous flow method for the synthesis of high-quality plasmonic single crystal silver nanocubes and nanospheres. The nanocubes were synthesized with controllable edge lengths from 20 to 48 nm. Single crystal nanospheres with a mean size of 29 nm were obtained by in-line continuous-flow etching of as-produced 39 nm nanocubes with an aqueous solution of FeNO3. In comparison to batch synthesis, the demonstrated processes represent highly scalable reactions, in terms of both production rate and endurance. The reactions were conducted in a commercially available flow-reactor system that is easily adaptable to industrial-scale production, facilitating widespread utilization of the procedure and the resulting nanoparticles. This journal is © The Royal Society of Chemistry 2013.

Catalyst retention in continuous flow with supercritical carbon dioxide

NARCIS (Netherlands)

Stouten, S.C.; Noel, T.; Wang, Q.; Hessel, V.

2014-01-01

This review discusses the retention of organometallic catalysts in continuous flow processes utilizing supercritical carbon dioxide. Due to its innovative properties, supercritical carbon dioxide offers interesting possibilities for process intensification. As a result of safety and cost

A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

Science.gov (United States)

Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

2017-10-01

In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

Science.gov (United States)

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-01-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

Continuous microwave flow synthesis of mesoporous hydroxyapatite.

Science.gov (United States)

Akram, Muhammad; Alshemary, Ammar Z; Goh, Yi-Fan; Wan Ibrahim, Wan Aini; Lintang, Hendrik O; Hussain, Rafaqat

2015-11-01

We have successfully used continuous microwave flow synthesis (CMFS) technique for the template free synthesis of mesoporous hydroxyapatite. The continuous microwave flow reactor consisted of a modified 2.45GHz household microwave, peristaltic pumps and a Teflon coil. This cost effective and efficient system was exploited to produce semi-crystalline phase pure nano-sized hydroxyapatite. Effect of microwave power, retention time and the concentration of reactants on the phase purity, degree of crystallinity and surface area of the final product was studied in detail. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were used to study the phase purity and composition of the product, while transmission electron microscopy (TEM) was used to study the effect of process parameters on the morphology of hydroxyapatite. The TEM analysis confirmed the formation of spherical particles at low microwave power; however the morphology of the particles changed to mesoporous needle and rod-like structure upon exposing the reaction mixture to higher microwave power and longer retention time inside the microwave. The in-vitro ion dissolution behavior of the as synthesized hydroxyapatite was studied by determining the amount of Ca(2+) ion released in SBF solution. Copyright © 2015 Elsevier B.V. All rights reserved.

Advanced Continuous Flow Platform for On-Demand Pharmaceutical Manufacturing.

Science.gov (United States)

Zhang, Ping; Weeranoppanant, Nopphon; Thomas, Dale A; Tahara, Kohei; Stelzer, Torsten; Russell, Mary Grace; O'Mahony, Marcus; Myerson, Allan S; Lin, Hongkun; Kelly, Liam P; Jensen, Klavs F; Jamison, Timothy F; Dai, Chunhui; Cui, Yuqing; Briggs, Naomi; Beingessner, Rachel L; Adamo, Andrea

2018-02-21

As a demonstration of an alternative to the challenges faced with batch pharmaceutical manufacturing including the large production footprint and lengthy time-scale, we previously reported a refrigerator-sized continuous flow system for the on-demand production of essential medicines. Building on this technology, herein we report a second-generation, reconfigurable and 25 % smaller (by volume) continuous flow pharmaceutical manufacturing platform featuring advances in reaction and purification equipment. Consisting of two compact [0.7 (L)×0.5 (D)×1.3 m (H)] stand-alone units for synthesis and purification/formulation processes, the capabilities of this automated system are demonstrated with the synthesis of nicardipine hydrochloride and the production of concentrated liquid doses of ciprofloxacin hydrochloride, neostigmine methylsulfate and rufinamide that meet US Pharmacopeia standards. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

DEFF Research Database (Denmark)

Rimhanen-Finne, R.; Enemark, Heidi L.; Kolehmainen, J.

2007-01-01

The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5) oocysts/g, while the detection limit for Giardia ELISA was 10(4) cysts/g. The Cryptosporidium ELISA showed 94% specificity...... zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs...

Isothermal Amplification and Lateral-Flow Assay for Detecting Crown-Gall-Causing Agrobacterium spp.

Science.gov (United States)

Fuller, Skylar L; Savory, Elizabeth A; Weisberg, Alexandra J; Buser, Jessica Z; Gordon, Michael I; Putnam, Melodie L; Chang, Jeff H

2017-09-01

Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

Evaluation of Chicken IgY Generated Against Canine Parvovirus Viral-Like Particles and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Assay for Canine Parvovirus Detection.

Science.gov (United States)

He, Jinxin; Wang, Yuan; Sun, Shiqi; Zhang, Xiaoying

2015-11-01

Immunoglobulin Y (IgY) antibodies were generated against canine parvovirus virus-like particles (CPV-VLPs) antigen using chickens. Anti-CPV-VLPs-IgY was extracted from hen egg yolk and used for developing enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for the detection of CPV in dog feces. The cutoff negative values for anti-CPV-VLPs-IgY were determined using negative fecal samples (already confirmed by polymerase chain reaction [PCR]). In both ELISA and ICA, there was no cross-reaction with other diarrheal pathogens. Thirty-four fecal samples were collected from dogs with diarrhea, of which 26.47% were confirmed as CPV-positive samples by PCR, while 29.41% and 32.35% of the samples were found to be positive by ELISA and ICA, respectively. The developed ELISA and ICA exhibited 97.06% and 94.12% conformity with PCR. Higher sensitivity and specificity were observed for IgY-based ELISA and ICA. Thus, they could be suitable for routine use in the diagnosis of CPV in dogs.

COST-EFFECTIVENESS OF CONTINUOUS-FLOW LEFT VENTRICULAR ASSIST DEVICES

NARCIS (Netherlands)

Neyt, Mattias; Van den Bruel, Ann; Smit, Yolba; De Jonge, Nicolaas; Erasmus, Michiel; Van Dijk, Diederik; Vlayen, Joan

Objectives: Mechanical circulatory support through left ventricular assist devices (LVADs) improves survival and quality of life for patients with end-stage heart failure who are ineligible for cardiac transplantation. Our aim was to calculate the cost-effectiveness of continuous-flow LVADs.

Dynamic Transitions and Baroclinic Instability for 3D Continuously Stratified Boussinesq Flows

Science.gov (United States)

Şengül, Taylan; Wang, Shouhong

2018-02-01

The main objective of this article is to study the nonlinear stability and dynamic transitions of the basic (zonal) shear flows for the three-dimensional continuously stratified rotating Boussinesq model. The model equations are fundamental equations in geophysical fluid dynamics, and dynamics associated with their basic zonal shear flows play a crucial role in understanding many important geophysical fluid dynamical processes, such as the meridional overturning oceanic circulation and the geophysical baroclinic instability. In this paper, first we derive a threshold for the energy stability of the basic shear flow, and obtain a criterion for local nonlinear stability in terms of the critical horizontal wavenumbers and the system parameters such as the Froude number, the Rossby number, the Prandtl number and the strength of the shear flow. Next, we demonstrate that the system always undergoes a dynamic transition from the basic shear flow to either a spatiotemporal oscillatory pattern or circle of steady states, as the shear strength of the basic flow crosses a critical threshold. Also, we show that the dynamic transition can be either continuous or catastrophic, and is dictated by the sign of a transition number, fully characterizing the nonlinear interactions of different modes. Both the critical shear strength and the transition number are functions of the system parameters. A systematic numerical method is carried out to explore transition in different flow parameter regimes. In particular, our numerical investigations show the existence of a hypersurface which separates the parameter space into regions where the basic shear flow is stable and unstable. Numerical investigations also yield that the selection of horizontal wave indices is determined only by the aspect ratio of the box. We find that the system admits only critical eigenmodes with roll patterns aligned with the x-axis. Furthermore, numerically we encountered continuous transitions to multiple

Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-Linked Immunosorbent Assay

Directory of Open Access Journals (Sweden)

Nischay Mishra

2018-03-01

Full Text Available Zika virus (ZIKV is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0% and specificity (95.9% versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

Science.gov (United States)

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

2014-12-01

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

FEATURES OF LONG-TERM MECHANICAL CIRCULATORY SUPPORT WITH CONTINUOUS-FLOW PUMP

Directory of Open Access Journals (Sweden)

G. P. Itkin

2012-01-01

Full Text Available In a review of the comparative analysis of methods and tools for long-term mechanical circulatory support with continuous flow and pulsatile flow implantable pumps. Particular attention is paid to the choice of the optimal modes of the operation of pumps based on the physical principles of the interaction between a the steady flow of blood to the pulsatile mechanics of the heart chambers. 

Continuous infusion thermodilution for assessment of coronary flow: Theoretical background and in vitro validation

NARCIS (Netherlands)

Veer, van 't M.; Geven, M.C.F.; Rutten, M.C.M.; Horst, van der A.; Aarnoudse, W.H.; Pijls, N.H.J.; Vosse, van de F.N.

2009-01-01

Direct volumetric assessment of coronary flow during cardiac catheterization has not been available so far. In the current study continuous infusion thermodilution, a method based on continuous infusion of saline into a selective coronary artery is evaluated. Theoretically, volumetric flow can be

The use of enzyme-linked immunosorbent assay systems for the serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. Groen (Jan); H.F. Egberink (Herman); G.H.A. Borst (Gerrit); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractComplex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-,

Continuous manufacturing of active pharmaceutical ingredients via flow technology

NARCIS (Netherlands)

Borukhova, S.; Hessel, V.; Kleinbudde, P.; Khinast, J.; Rantanen, J.

2017-01-01

The main drivers to implement continuous manufacturing are aspects related to logistics, quality of the final product, chemistry to be implemented, process and safety concerns. Flow technology offers a platform to realize those drivers. This chapter introduces the reader to a relatively new

Field assessment of the enzyme linked immunosorbent assay for foot-and-mouth disease virus diagnosis and typing

International Nuclear Information System (INIS)

Smitsaart, E.; Fondevila, N.; Compaired, D.; Maradei, E.; Fernandez, E.

1998-01-01

The objective of the present study was to evaluate the enzyme linked immunosorbent assay (ELISA) in comparison with the complement fixation test (CFT) for the diagnosis and typing of foot-and-mouth disease (FMD) virus (FMDV). Diagnostic material was epithelium from either suspected cases of FMD or from animals experimentally infected with FMDV. Epithelial suspensions and supernatant fluids from cell culture passage were assayed by CFT and ELISA. The superiority of the ELISA over the CFT was demonstrated: 1) the detection rate was 23% higher than that of CFT on original (epithelial) suspensions (OS) submissions of all sample (positive and negative) and 30% higher on supernatant fluids from cell culture passage, 2) the detection rate of ELISA on OS of confirmed positive samples was 28% higher than that of CFT, 3) no significant differences were observed in the detection and typing rates between the PANAFTOSA and FAO/IAEA ELISA kits (P<0.05) and 4) the sensitivity of the ELISA was 16 to 85 times higher than that of CFT when serial dilutions of sample homogenates were examined. (author)

Improved Peak Capacity for Capillary Electrophoretic Separations of Enzyme Inhibitors with Activity-Based Detection Using Magnetic Bead Microreactors

Science.gov (United States)

Yan, Xiaoyan; Gilman, S. Douglass

2010-01-01

A technique for separating and detecting enzyme inhibitors was developed using capillary electrophoresis with an enzyme microreactor. The on-column enzyme microreactor was constructed using NdFeB magnet(s) to immobilize alkaline phosphatase-coated superparamagnetic beads (2.8 μm diameter) inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with the substrate, AttoPhos. Product generated in the enzyme microreactor was detected by laser-induced fluorescence. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due to decreased product formation. The goal of this study was to improve peak capacities for inhibitor separations relative to previous work, which combined continuous engagement electrophoretically mediated microanalysis (EMMA) and transient engagement EMMA to study enzyme inhibition. The effects of electric field strength, bead injection time and inhibitor concentrations on peak capacity and peak width were investigated. Peak capacities were increased to ≥20 under optimal conditions of electric field strength and bead injection time for inhibition assays with arsenate and theophylline. Five reversible inhibitors of alkaline phosphatase (theophylline, vanadate, arsenate, L-tryptophan and tungstate) were separated and detected to demonstrate the ability of this technique to analyze complex inhibitor mixtures. PMID:20024913

Correlation between ELISA and ML Flow assays applied to 60 Brazilian patients affected by leprosy

NARCIS (Netherlands)

Da Silva, Rozana C.; Lyon, Sandra; Lyon, Ana C.; Grossi, Maria A. F.; Lyon, Silvia H.; Bührer-Sékula, Samira; Antunes, Carlos M. F.

2010-01-01

Serological tests can be helpful in classifying leprosy patients as having either the paucibacillary or the multibacillary form. The aim of this study was to evaluate the concordance between two serological assays, i.e. ML Flow and ELISA, in a population of leprosy patients in Brazil. The

Development of Two Antibody Detection Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Human Chronic Fascioliasis

Science.gov (United States)

Cabán-Hernández, Kimberly; Gaudier, José F.; Ruiz-Jiménez, Caleb

2014-01-01

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories. PMID:24353000

Development of two antibody detection enzyme-linked immunosorbent assays for serodiagnosis of human chronic fascioliasis.

Science.gov (United States)

Cabán-Hernández, Kimberly; Gaudier, José F; Ruiz-Jiménez, Caleb; Espino, Ana M

2014-03-01

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories.

Screening of Enzyme Biomarker for Nanotoxicity of Zinc Oxide in OREOCHROMIS MOSSAMBICUS

Science.gov (United States)

Subramanian, Periasamy; Bupesh, Giridharan

2011-06-01

Experiments were conducted to determine the effects of Zinc oxide (ZnO) nanoparticles (NPs) on fish models. Oreochromis mossambicus was orally administered with ZnO NPs (50-100 nm) once and its effects at five different concentrations (60 ppm-100 ppm) were observed for 12 days. Enzymatic assays were performed at every three days interval in the vital tissues of liver, gill, muscle and kidney. The defense enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S transferase (GST) exerted a dose dependent elevation up to 6 days. This hike then declines in higher concentrations and extended duration. Whereas the tissue damaging enzymes, glutamate oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) exhibited a dose and duration dependent increase until the end of the experiment. Among these enzymes, the antioxidant enzymes response to ZnO NP toxicity on fish showed notable continuous induction. This study demonstrates that antioxidant enzymes responses in O. mossambicus could be used as a biomarker for the early detection of nanotoxicity.

Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b

International Nuclear Information System (INIS)

Kristensen, K.; Weis Bentzon, M.

1992-01-01

The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was indepenedent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines. (au)

Solar oxidation and removal of arsenic--Key parameters for continuous flow applications.

Science.gov (United States)

Gill, L W; O'Farrell, C

2015-12-01

Solar oxidation to remove arsenic from water has previously been investigated as a batch process. This research has investigated the kinetic parameters for the design of a continuous flow solar reactor to remove arsenic from contaminated groundwater supplies. Continuous flow recirculated batch experiments were carried out under artificial UV light to investigate the effect of different parameters on arsenic removal efficiency. Inlet water arsenic concentrations of up to 1000 μg/L were reduced to below 10 μg/L requiring 12 mg/L iron after receiving 12 kJUV/L radiation. Citrate however was somewhat surprisingly found to promote a detrimental effect on the removal process in the continuous flow reactor studies which is contrary to results found in batch scale tests. The impact of other typical water groundwater quality parameters (phosphate and silica) on the process due to their competition with arsenic for photooxidation products revealed a much higher sensitivity to phosphate ions compared to silicate. Other results showed no benefit from the addition of TiO2 photocatalyst but enhanced arsenic removal at higher temperatures up to 40 °C. Overall, these results have indicated the kinetic envelope from which a continuous flow SORAS single pass system could be more confidently designed for a full-scale community groundwater application at a village level. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

Science.gov (United States)

Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

2002-04-01

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay

Semi-continuous protein fractionating using affinity cross-flow filtration

NARCIS (Netherlands)

Borneman, Zandrie; Zhang, W.; van den Boomgaard, Anthonie; Smolders, C.A.

2002-01-01

Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This

An enzyme-linked immunosorbent assay and a gold-nanoparticle based immuno chromatographic test for amatoxins using recombinant antibody

International Nuclear Information System (INIS)

He, Kuo; Zhao, Ruiping; Wang, Lixia; Feng, Tingting; Wei, Dong; Zhang, Xiuyuan

2016-01-01

The authors describe two kinds of rapid assays for the determination of amatoxins in mushrooms. The first is an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase. The second is a rapid immuno chromatographic assay that uses colloidal gold as a red label (CG-ICA). Both are based on the use of a well-characterized recombinant single chain variable fragment antibody (named scFv-A4). The half-maximum inhibition concentrations (IC50) of α-amanitin, β-amanitin and γ-amanitin are 78, 85 and 90 ng⋅mL"-"1, and the limits of detection (LODs; for IC15) are 1.9, 2.1 and 2.8 ng⋅mL"-"1. The method was applied to the determination of amanitins in mushrooms, and the LODs for α-amanitin, β-amanitin and γ-amanitin in mushroom samples were found to be 4.9, 6.4 and 8.3 ng⋅mL"-"1. The visual minimum detection limits of the optimized CGIA are 4 and 6 ng⋅mL"-"1 for mushroom samples. The test can be performed within 10 min. The results of the analysis of spiked samples showed that the CG-IA can rapidly and semi-quantitatively quantify amatoxins in mushroom samples on site and at low costs. (author)

Renormalization group flows and continual Lie algebras

International Nuclear Information System (INIS)

Bakas, Ioannis

2003-01-01

We study the renormalization group flows of two-dimensional metrics in sigma models using the one-loop beta functions, and demonstrate that they provide a continual analogue of the Toda field equations in conformally flat coordinates. In this algebraic setting, the logarithm of the world-sheet length scale, t, is interpreted as Dynkin parameter on the root system of a novel continual Lie algebra, denoted by (d/dt;1), with anti-symmetric Cartan kernel K(t,t') = δ'(t-t'); as such, it coincides with the Cartan matrix of the superalgebra sl(N vertical bar N+1) in the large-N limit. The resulting Toda field equation is a non-linear generalization of the heat equation, which is integrable in target space and shares the same dissipative properties in time, t. We provide the general solution of the renormalization group flows in terms of free fields, via Baecklund transformations, and present some simple examples that illustrate the validity of their formal power series expansion in terms of algebraic data. We study in detail the sausage model that arises as geometric deformation of the O(3) sigma model, and give a new interpretation to its ultra-violet limit by gluing together two copies of Witten's two-dimensional black hole in the asymptotic region. We also provide some new solutions that describe the renormalization group flow of negatively curved spaces in different patches, which look like a cane in the infra-red region. Finally, we revisit the transition of a flat cone C/Z n to the plane, as another special solution, and note that tachyon condensation in closed string theory exhibits a hidden relation to the infinite dimensional algebra (d/dt;1) in the regime of gravity. Its exponential growth holds the key for the construction of conserved currents and their systematic interpretation in string theory, but they still remain unknown. (author)

Detection of Shiga Toxins by Lateral Flow Assay

Directory of Open Access Journals (Sweden)

Kathryn H. Ching

2015-04-01

Full Text Available Shiga toxin-producing Escherichia coli (STEC produce shiga toxins (Stxs that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

Continuous Flow Science in an Undergraduate Teaching Laboratory: Photocatalytic Thiol-Ene Reaction Using Visible Light

Science.gov (United States)

Santandrea, Jeffrey; Kairouz, Vanessa; Collins, Shawn K.

2018-01-01

An undergraduate teaching laboratory experiment involving a continuous flow, photocatalytic thiol-ene reaction using visible-light irradiation is described that allows students to explore concepts of green chemistry, photochemistry, photocatalysis, and continuous flow chemistry.

Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis

NARCIS (Netherlands)

Bokma, Evert; Barends, Thomas; Terwisscha van Scheltinga, Anke C.; Dijkstra, Bauke W.; Beintema, Jaap J.

2000-01-01

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore, Products mere separated by HPLC and the amount of product was

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

Science.gov (United States)

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-06-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

Continuous production of lipase-catalyzed biodiesel in a packed-bed reactor: optimization and enzyme reuse study.

Science.gov (United States)

Chen, Hsiao-Ching; Ju, Hen-Yi; Wu, Tsung-Ta; Liu, Yung-Chuan; Lee, Chih-Chen; Chang, Cheng; Chung, Yi-Lin; Shieh, Chwen-Jen

2011-01-01

An optimal continuous production of biodiesel by methanolysis of soybean oil in a packed-bed reactor was developed using immobilized lipase (Novozym 435) as a catalyst in a tert-butanol solvent system. Response surface methodology (RSM) and Box-Behnken design were employed to evaluate the effects of reaction temperature, flow rate, and substrate molar ratio on the molar conversion of biodiesel. The results showed that flow rate and temperature have significant effects on the percentage of molar conversion. On the basis of ridge max analysis, the optimum conditions were as follows: flow rate 0.1 mL/min, temperature 52.1°C, and substrate molar ratio 1 : 4. The predicted and experimental values of molar conversion were 83.31 ± 2.07% and 82.81 ± .98%, respectively. Furthermore, the continuous process over 30 days showed no appreciable decrease in the molar conversion. The paper demonstrates the applicability of using immobilized lipase and a packed-bed reactor for continuous biodiesel synthesis.

Continuous flow photolysis of aryl azides: Preparation of 3H-azepinones

Directory of Open Access Journals (Sweden)

Farhan R. Bou-Hamdan

2011-08-01

Full Text Available Photolysis of aryl azides to give nitrenes, and their subsequent rearrangement in the presence of water to give 3H-azepinones, is performed in continuous flow in a photoreactor constructed of fluorinated ethylene polymer (FEP tubing. Fine tuning of the reaction conditions using the flow reactor allowed minimization of secondary photochemical reactions.

Continuous flow chemistry: a discovery tool for new chemical reactivity patterns.

Science.gov (United States)

Hartwig, Jan; Metternich, Jan B; Nikbin, Nikzad; Kirschning, Andreas; Ley, Steven V

2014-06-14

Continuous flow chemistry as a process intensification tool is well known. However, its ability to enable chemists to perform reactions which are not possible in batch is less well studied or understood. Here we present an example, where a new reactivity pattern and extended reaction scope has been achieved by transferring a reaction from batch mode to flow. This new reactivity can be explained by suppressing back mixing and precise control of temperature in a flow reactor set up.

Continuous flow chemistry: a discovery tool for new chemical reactivity patterns

OpenAIRE

Hartwig, Jan; Metternich, Jan B.; Nikbin, Nikzad; Kirschning, Andreas; Ley, Steven V.

2014-01-01

Continuous flow chemistry as a process intensification tool is well known. However, its ability to enable chemists to perform reactions which are not possible in batch is less well studied or understood. Here we present an example, where a new reactivity pattern and extended reaction scope has been achieved by transferring a reaction from batch mode to flow. This new reactivity can be explained by suppressing back mixing and precise control of temperature in a flow reactor set up.

A Dual Electrochemical Sensor Based on a Test-strip Assay for the Quantitative Determination of Albumin and Creatinine.

Science.gov (United States)

Yasukawa, Tomoyuki; Kiba, Yuya; Mizutani, Fumio

2015-01-01

A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6(3-) to Fe(CN)6(4-), which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL(-1), respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange.

Science.gov (United States)

Curtis, C F

2001-02-24

This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable.

Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

2015-01-01

Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL −1 with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO 2 particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors

Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

Energy Technology Data Exchange (ETDEWEB)

Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

2015-08-05

Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

Science.gov (United States)

Li, Zhi; Zhang, Yanlong; Wang, Huiguo; Jin, Jinhua; Li, Wenzhe

2013-01-01

A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease. PMID:24124274

Design considerations of a hollow microneedle-optofluidic biosensing platform incorporating enzyme-linked assays

Science.gov (United States)

Ranamukhaarachchi, Sahan A.; Padeste, Celestino; Häfeli, Urs O.; Stoeber, Boris; Cadarso, Victor J.

2018-02-01

A hollow metallic microneedle is integrated with microfluidics and photonic components to form a microneedle-optofluidic biosensor suitable for therapeutic drug monitoring (TDM) in biological fluids, like interstitial fluid, that can be collected in a painless and minimally-invasive manner. The microneedle inner lumen surface is bio-functionalized to trap and bind target analytes on-site in a sample volume as small as 0.6 nl, and houses an enzyme-linked assay on its 0.06 mm2 wall. The optofluidic components are designed to rapidly quantify target analytes present in the sample and collected in the microneedle using a simple and sensitive absorbance scheme. This contribution describes how the biosensor components were optimized to detect in vitro streptavidin-horseradish peroxidase (Sav-HRP) as a model analyte over a large detection range (0-7.21 µM) and a very low limit of detection (60.2 nM). This biosensor utilizes the lowest analyte volume reported for TDM with microneedle technology, and presents significant avenues to improve current TDM methods for patients, by potentially eliminating blood draws for several drug candidates.

Direct immunofluorescence and enzyme-linked immunosorbent assays for evaluating chlorinated hydrocarbon degrading bacteria

Energy Technology Data Exchange (ETDEWEB)

Brigmon, R.L.; Franck, M.M.; Brey, J.; Fliermans, C.B. [Westinghouse Savannah River, Aiken, SC (United States). Environmental Biotechnology Section; Scott, D.; Lanclos, K. [Medical Coll. of Georgia, Augusta, GA (United States)

1997-06-01

Immunological procedures were developed to enumerate chlorinated hydrocarbon degrading bacteria. Polyclonal antibodies (Pabs) were produced by immunizing New Zealand white rabbits against 18 contaminant-degrading bacteria. These included methanotrophic and chlorobenzene (CB) degrading species. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Pabs. Direct fluorescent antibodies (DFAs) were developed with these Pabs against select methanotrophic bacteria isolated from a trichloroethylene (TCE) contaminated landfill at the Savannah River Site (SRS) and cultures from the American Type Culture Collection (ATCC). Analysis of cross reactivity testing data showed some of the Pabs to be group specific while others were species specific. The threshold of sensitivity for the ELISA is 105 bacteria cells/ml. The DFA can detect as few as one bacterium per ml after concentration. Results from the DFA and ELISA techniques for enumeration of methanotrophic bacteria in groundwater were higher but not significantly different (P < 0.05) compared to indirect microbiological techniques such as MPN. These methods provide useful information on in situ community structure and function for bioremediation applications within 1--4 hours of sampling.

A miniature CSTR cascade for continuous flow of reactions containing solids

OpenAIRE

Mo, Yiming; Jensen, Klavs F

2016-01-01

Continuous handling of solids creates challenges for realizing continuous production of pharmaceuticals and fine chemicals. We present a new miniature continuous stirred-tank reactor (CSTR) cascade to handle solid-forming reactions in flow. Single-phase residence time distribution (RTD) measurements of the CSTR cascade reveal nearly ideal CSTR mixing behavior of the individual units. Consistency of experimental and predicted conversions of a Diels–Alder reaction further confirms the CSTR perf...

Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Oviedo, J M; Valiño, F; Plasencia, I

2001-01-01

We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...... used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein...

Biocatalytic synthesis of the Green Note trans-2-hexenal in a continuous-flow microreactor.

Science.gov (United States)

van Schie, Morten M C H; Pedroso de Almeida, Tiago; Laudadio, Gabriele; Tieves, Florian; Fernández-Fueyo, Elena; Noël, Timothy; Arends, Isabel W C E; Hollmann, Frank

2018-01-01

The biocatalytic preparation of trans -hex-2-enal from trans -hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii ( Pe AAOx) is reported. As O 2 -dependent enzyme Pe AAOx-dependent reactions are generally plagued by the poor solubility of O 2 in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s -1 ) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.

Angiotensin-I-converting enzyme and gallium scan in noninvasive evaluation of sarcoidosis

Energy Technology Data Exchange (ETDEWEB)

Nosal, A. (Harbor General Hospital, Torrance, CA); Schleissner, L.A.; Mishkin, F.S.; Lieberman, J.

1979-03-01

Angiotensin-converting enzyme assays and gallium-scan results were obtained from 27 patients with biopsy-proven, clinically active sarcoidosis. Twenty-three of these patients had elevated converting enzyme levels, and 22 had positive gallium-scan results. Three of four patients with normal or borderline-elevated levels of angiotensin-converting enzyme also had positive gallium-scan results. Of 156 nonsarcoid patients (pulmonary and other diseases), 27 were found to have elevated serum converting enzyme levels, and 25 of these had negative gallium-scan results. These results indicate that the combination of an assay of angiotensin-converting enzyme and gallium scan increases diagnostic specificity from 83% to 99% without sacrificing sensitivity. It was concluded that the concurrent use of angiotensin-converting enzyme assay and gallium scan is of value in the diagnosis of sarcoidosis.

Angiotensin-I-converting enzyme and gallium scan in noninvasive evaluation of sarcoidosis

International Nuclear Information System (INIS)

Nosal, A.; Schleissner, L.A.; Mishkin, F.S.; Lieberman, J.

1979-01-01

Angiotensin-converting enzyme assays and gallium-scan results were obtained from 27 patients with biopsy-proven, clinically active sarcoidosis. Twenty-three of these patients had elevated converting enzyme levels, and 22 had positive gallium-scan results. Three of four patients with normal or borderline-elevated levels of angiotensin-converting enzyme also had positive gallium-scan results. Of 156 nonsarcoid patients (pulmonary and other diseases), 27 were found to have elevated serum converting enzyme levels, and 25 of these had negative gallium-scan results. These results indicate that the combination of an assay of angiotensin-converting enzyme and gallium scan increases diagnostic specificity from 83% to 99% without sacrificing sensitivity. It was concluded that the concurrent use of angiotensin-converting enzyme assay and gallium scan is of value in the diagnosis of sarcoidosis

Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody▿

OpenAIRE

He, Qigai; Velumani, Sumathy; Du, Qingyun; Lim, Chee Wee; Ng, Fook Kheong; Donis, Ruben; Kwang, Jimmy

2007-01-01

The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (H...

Verification of a computational cardiovascular system model comparing the hemodynamics of a continuous flow to a synchronous valveless pulsatile flow left ventricular assist device.

Science.gov (United States)

Gohean, Jeffrey R; George, Mitchell J; Pate, Thomas D; Kurusz, Mark; Longoria, Raul G; Smalling, Richard W

2013-01-01

The purpose of this investigation is to use a computational model to compare a synchronized valveless pulsatile left ventricular assist device with continuous flow left ventricular assist devices at the same level of device flow, and to verify the model with in vivo porcine data. A dynamic system model of the human cardiovascular system was developed to simulate the support of a healthy or failing native heart from a continuous flow left ventricular assist device or a synchronous pulsatile valveless dual-piston positive displacement pump. These results were compared with measurements made during in vivo porcine experiments. Results from the simulation model and from the in vivo counterpart show that the pulsatile pump provides higher cardiac output, left ventricular unloading, cardiac pulsatility, and aortic valve flow as compared with the continuous flow model at the same level of support. The dynamic system model developed for this investigation can effectively simulate human cardiovascular support by a synchronous pulsatile or continuous flow ventricular assist device.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

DEFF Research Database (Denmark)

Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

2017-01-01

to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

Renewable Wood Pulp Paper Reactor with Hierarchical Micro/Nanopores for Continuous-Flow Nanocatalysis.

Science.gov (United States)

Koga, Hirotaka; Namba, Naoko; Takahashi, Tsukasa; Nogi, Masaya; Nishina, Yuta

2017-06-22

Continuous-flow nanocatalysis based on metal nanoparticle catalyst-anchored flow reactors has recently provided an excellent platform for effective chemical manufacturing. However, there has been limited progress in porous structure design and recycling systems for metal nanoparticle-anchored flow reactors to create more efficient and sustainable catalytic processes. In this study, traditional paper is used for a highly efficient, recyclable, and even renewable flow reactor by tailoring the ultrastructures of wood pulp. The "paper reactor" offers hierarchically interconnected micro- and nanoscale pores, which can act as convective-flow and rapid-diffusion channels, respectively, for efficient access of reactants to metal nanoparticle catalysts. In continuous-flow, aqueous, room-temperature catalytic reduction of 4-nitrophenol to 4-aminophenol, a gold nanoparticle (AuNP)-anchored paper reactor with hierarchical micro/nanopores provided higher reaction efficiency than state-of-the-art AuNP-anchored flow reactors. Inspired by traditional paper materials, successful recycling and renewal of AuNP-anchored paper reactors were also demonstrated while high reaction efficiency was maintained. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Applying lean principles to achieve continuous flow in 3PLs’ outbound processes

NARCIS (Netherlands)

Overboom, M.A.; Small, J.S.; Naus, A.J.A.M.; de Haan, J.A.C.

2013-01-01

The article offers information on the application of lean principles to achieve continuous flow in third party logistics providers (3PLs). It mentions that lean management principles and practices have been traditionally applied to manufacturing systems and try to make products flow through the

Identification of a vesicular-arbuscular mycorrhizal fungus by using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Science.gov (United States)

Wright, S F; Morton, J B; Sworobuk, J E

1987-09-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

Voltage stability analysis using a modified continuation load flow ...

African Journals Online (AJOL)

This paper addresses the rising problem of identifying the voltage stability limits of load buses in a power system and how to optimally place capacitor banks for voltage stability improvement. This paper uses the concept of the continuation power flow analysis used in voltage stability analysis. It uses the modified ...

Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

Directory of Open Access Journals (Sweden)

Marco Tamba

2010-01-01

Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

Comparison of enzyme-linked immunosorbent assay and rapid chemiluminescent analyser in the detection of myeloperoxidase and proteinase 3 autoantibodies.

Science.gov (United States)

Pucar, Phillippa A; Hawkins, Carolyn A; Randall, Katrina L; Li, Candice; McNaughton, Euan; Cook, Matthew C

2017-06-01

Antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3) are vital in the diagnosis and management of ANCA-associated vasculitis. A chemiluminescent immunoassay (CLIA; Quanta Flash) provides MPO and PR3 antibody results in 30 minutes, which is much faster than enzyme-linked immunosorbent assay (ELISA). We compared the performance of ELISA (Orgentec) and CLIA (Quanta Flash) for MPO and PR3 antibody quantitation on 303 samples, comprising 196 consecutive samples received in a single diagnostic laboratory over a 3 month period, and 107 samples collected from 42 known vasculitis patients over a 40 month period. We observed a correlation between both methods using spearman correlation coefficients (MPO, r s  = 0.63, p assays) and disease relapse (correlation for both MPO and PR3 antibody quantitation r s  = 0.84, p = 0.03 and r s  = 0.78, p ELISA for measurement of MPO and PR3 antibodies. Copyright © 2017. Published by Elsevier B.V.

An enzyme-linked immunosorbent assay for monoester-type aconitic alkaloids and its application in the pharmacokinetic study of benzoylhypaconine in rats.

Science.gov (United States)

Liu, Can-Can; Xu, Yun-Hui; Yuan, Shuai; Xu, Yu; Hua, Mo-Li

2018-04-01

A new enzyme-linked immunosorbent assay (ELISA) method for quantitative determination of monoester-type aconitic alkaloids was developed. The antibodies derived from the immunogen of benzoylmesaconine (BM) could be electively affined to benzoylaconitine-type alkaloids with an ester bond (14-benzoyl-), especially to benzoylhypaconine (BH, 140.02% of cross-reactivity). The effective working range of BH was 1 ng/ml to 5 μg/ml; the lower limit of detection and the quantification were 0.35 and 0.97 ng/ml, respectively. The values of CV for intra-day and inter-day assays and recovery ratios were in acceptable ranges. The results of stability experiments were also satisfactory. This validated method was employed for pharmacokinetic study of BH in rats and the bioavailability orally administered was estimated to be 16.3%.

Enzyme-Linked Immunosorbent Assay Specific for (1→6) Branched, (1→3)-β-d-Glucan Detection in Environmental Samples

OpenAIRE

Milton, Donald K.; Alwis, K. Udeni; Fisette, Leslie; Muilenberg, Michael

2001-01-01

(1→3)-β-d-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1→6) branched, (1→3)-β-d-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1→3)-β-d-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall β-d-glucans...

One-step synthesis of pyridines and dihydropyridines in a continuous flow microwave reactor

Directory of Open Access Journals (Sweden)

Mark C. Bagley

2013-09-01

Full Text Available The Bohlmann–Rahtz pyridine synthesis and the Hantzsch dihydropyridine synthesis can be carried out in a microwave flow reactor or using a conductive heating flow platform for the continuous processing of material. In the Bohlmann–Rahtz reaction, the use of a Brønsted acid catalyst allows Michael addition and cyclodehydration to be carried out in a single step without isolation of intermediates to give the corresponding trisubstituted pyridine as a single regioisomer in good yield. Furthermore, 3-substituted propargyl aldehydes undergo Hantzsch dihydropyridine synthesis in preference to Bohlmann–Rahtz reaction in a very high yielding process that is readily transferred to continuous flow processing.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

Science.gov (United States)

Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

2014-12-01

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Continuous Production of Lipase-Catalyzed Biodiesel in a Packed-Bed Reactor: Optimization and Enzyme Reuse Study

Directory of Open Access Journals (Sweden)

Hsiao-Ching Chen

2011-01-01

Full Text Available An optimal continuous production of biodiesel by methanolysis of soybean oil in a packed-bed reactor was developed using immobilized lipase (Novozym 435 as a catalyst in a tert-butanol solvent system. Response surface methodology (RSM and Box-Behnken design were employed to evaluate the effects of reaction temperature, flow rate, and substrate molar ratio on the molar conversion of biodiesel. The results showed that flow rate and temperature have significant effects on the percentage of molar conversion. On the basis of ridge max analysis, the optimum conditions were as follows: flow rate 0.1 mL/min, temperature 52.1∘C, and substrate molar ratio 1 : 4. The predicted and experimental values of molar conversion were 83.31±2.07% and 82.81±.98%, respectively. Furthermore, the continuous process over 30 days showed no appreciable decrease in the molar conversion. The paper demonstrates the applicability of using immobilized lipase and a packed-bed reactor for continuous biodiesel synthesis.

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

Science.gov (United States)

Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

2013-01-01

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

DECREASE Final Technical Report: Development of a Commercial Ready Enzyme Application System for Ethanol

Energy Technology Data Exchange (ETDEWEB)

Teter, Sarah A

2012-04-18

.5 kg kettle reactors) was performed to compare the starting enzyme mixture CZP00005 with CTec3 alone; these results indicated a 1.9X dose- reduction for 80% conversion. The CTec3 composition does not include the best available enzyme components from the DECREASE effort. While these components are not yet available in a commercial product, experimental mixtures were assayed in a smaller scale assay using DECREASE PCS, at high solids loadings (21.5% TS). The results indicated that the newer mixtures required 2.9X-less enzyme for 90% conversion, and 3.2X-less enzyme for 80% conversion, relative to the starting enzyme cocktail. In conclusion, CTec3 delivers a 1.8-1.9X dose reduction on NREL PCS at high solids loadings, and the next generation enzyme from Novozymes will continue to show dramatically improved biochemical performance. CTec3 allows reduced costs today, and the experimental cocktails point to continued biotechnological improvements that will further drive down costs for biorefineries of tomorrow.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

Science.gov (United States)

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry.

Science.gov (United States)

Bayraç, Ceren; Öktem, Hüseyin Avni

2017-02-01

To monitor the specificity of Staphylococcus aureus aptamer (SA-31) against its target cell, we used enzyme-linked aptamer assay. In the presence of target cell, horseradish peroxidase-conjugated streptavidin bound to biotin-labeled SA-31 showed specific binding to S  aureus among 3 different bacteria with limit of detection of 10 3 colony-forming unit per milliliter. The apparent K a was 1.39 μM -1  ± 0.3 μM -1 . The binding of SA-31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K a , ΔH, and ΔG). Since binding of aptamer to its targets solely depends on its 3-dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA-31 to its target on surface of bacteria. At 4°C, SA-31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA-31 slightly varied from K a  = 1.56 μM -1  ± 0.69 μM -1 at 25°C to K a  = 1.03 μM -1  ± 0.9 μM -1 at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 μM -1  ± 0.38 μM -1 at 50mM, 1.60 μM -1  ± 0.11 μM -1 at 137mM, and 3.28 μM -1  ± 0.46 μM -1 at 200 mM of salt concentration. In this study, it was demonstrated that enzyme-linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus. Copyright © 2016 John Wiley & Sons, Ltd.

Immunological measurements on the disappearance of creatine kinase MM from the circulation. [Immunoradiometric assay

Energy Technology Data Exchange (ETDEWEB)

Wevers, R A; van Landeghem, A A.J.; Mul-Steinbusch, M W.F.J.; Bijdendijk, J G; Weerts, P; Kloeg, P; Soons, J B.J. [Rijksuniversiteit Utrecht (Netherlands)

1983-07-15

Both a two-site immunoradiometric assay and a two-site enzyme-linked immunosorbent assay for creatine kinase MM are described. Linearity, reproducibility and cross-reactivity of the assays are satisfactory. Creatine kinase MM incubated in a pH-controlled serum matrix loses its activity, but has its antigenic determinants affected as well. Of all the techniques used, only the immunoradiometric assay is capable of measuring part of the inactivated enzyme. Measurements with this assay on the sera of patients after a myocardial infarction show identical results for enzyme activity and creatine kinase protein quantity. The in vitro disappearance rate of creatine kinase activity is slow compared with the in vivo half-life of the enzyme. These two observations lead to the conclusion that creatine kinase is removed from the circulation long before it is inactivated in the blood stream.

Enzyme-linked immunosorbent assay (ELISA) for the detection of use of the synthetic cannabinoid agonists UR-144 and XLR-11 in human urine.

Science.gov (United States)

Mohr, Amanda L A; Ofsa, Bill; Keil, Alyssa Marie; Simon, John R; McMullin, Matthew; Logan, Barry K

2014-09-01

Ongoing changes in the synthetic cannabinoid drug market create the need for relevant targeted immunoassays for rapid screening of biological samples. We describe the validation and performance characteristics of an enzyme-linked immunosorbent assay designed to detect use of one of the most prevalent synthetic cannabinoids in urine, UR-144, by targeting its pentanoic acid metabolite. Fluorinated UR-144 (XLR-11) has been demonstrated to metabolize to this common product. The assay has significant cross-reactivity with UR-144-5-OH, UR-144-4-OH and XLR-11-4-OH metabolites, but assay's cutoff is 5 ng/mL relative to the pentanoic acid metabolite of UR-144, which is used as the calibrator. The method was validated with 90 positive and negative control urine samples for UR-144, XLR-11 and its metabolites tested versus liquid chromatography-tandem mass spectrometry. The accuracy, sensitivity and specificity were determined to be 100% for the assay at the specified cutoff. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Phenotypic and genotypic characterization of antioxidant enzyme system in human population exposed to radiation from mobile towers.

Science.gov (United States)

Gulati, Sachin; Yadav, Anita; Kumar, Neeraj; Priya, Kanu; Aggarwal, Neeraj K; Gupta, Ranjan

2018-03-01

In the present era, cellular phones have changed the life style of human beings completely and have become an essential part of their lives. The number of cell phones and cell towers are increasing in spite of their disadvantages. These cell towers transmit radiation continuously without any interruption, so people living within 100s of meters from the tower receive 10,000 to 10,000,000 times stronger signal than required for mobile communication. In the present study, we have examined superoxide dismutase (SOD) enzyme activity, catalase (CAT) enzyme activity, lipid peroxidation assay, and effect of functional polymorphism of SOD and CAT antioxidant genes against mobile tower-induced oxidative stress in human population. From our results, we have found a significantly lower mean value of manganese superoxide dismutase (MnSOD) enzyme activity, catalase (CAT) enzyme activity, and a high value of lipid peroxidation assay in exposed as compared to control subjects. Polymorphisms in antioxidant MnSOD and CAT genes significantly contributed to its phenotype. In the current study, a significant association of genetic polymorphism of antioxidant genes with genetic damage has been observed in human population exposed to radiations emitted from mobile towers.

A quantitative comet infection assay for influenza virus

Science.gov (United States)

Lindsay, Stephen M.; Timm, Andrea; Yin, John

2011-01-01

Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

Science.gov (United States)

Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

2014-03-03

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

Continuous-flow sheathless diamagnetic particle separation in ferrofluids

International Nuclear Information System (INIS)

Zhou, Yilong; Song, Le; Yu, Liandong; Xuan, Xiangchun

2016-01-01

Separating particles from a complex mixture is often necessary in many chemical and biomedical applications. This work presents a continuous-flow sheathless diamagnetic particle separation in ferrofluids through U-shaped microchannels. Due to the action of a size-dependent magnetic force, diamagnetic particles are focused into a single stream in the inlet branch of the U-turn and then continuously separated into two streams in its outlet branch. A 3D numerical model is developed to predict and understand the diamagnetic particle transport during this separation process. The numerical predictions are found to agree well with the experimental observations in a systematic study of the effects of multiple parameters including ferrofluid flow rate, concentration and magnet-channel distance. Additional numerical studies of the geometric effects of the U-turn reveal that increasing the outlet-branch width of the U-turn can significantly enhance the diamagnetic particle separation in ferrofluids. - Highlights: • Particles are focused and separated in the two branches of a U-shaped microchannel. • Negative magnetophoretic motion in ferrofluids causes the particle deflection. • A 3D numerical model is developed to simulate the particle separation. • Multiple parametric effects are studied both experimentally and numerically. • Increasing the outlet-branch width significantly enhances the particle separation.

Continuous-flow sheathless diamagnetic particle separation in ferrofluids

Energy Technology Data Exchange (ETDEWEB)

Zhou, Yilong [Department of Mechanical Engineering, Clemson University, Clemson, SC 29634-0921 (United States); Song, Le [School of Instrument Science and Opto-electronic Engineering, Hefei University of Technology, Hefei 230009 (China); Yu, Liandong, E-mail: liandongyu@hfut.edu.cn [School of Instrument Science and Opto-electronic Engineering, Hefei University of Technology, Hefei 230009 (China); Xuan, Xiangchun, E-mail: xcxuan@clemson.edu [Department of Mechanical Engineering, Clemson University, Clemson, SC 29634-0921 (United States)

2016-08-15

Separating particles from a complex mixture is often necessary in many chemical and biomedical applications. This work presents a continuous-flow sheathless diamagnetic particle separation in ferrofluids through U-shaped microchannels. Due to the action of a size-dependent magnetic force, diamagnetic particles are focused into a single stream in the inlet branch of the U-turn and then continuously separated into two streams in its outlet branch. A 3D numerical model is developed to predict and understand the diamagnetic particle transport during this separation process. The numerical predictions are found to agree well with the experimental observations in a systematic study of the effects of multiple parameters including ferrofluid flow rate, concentration and magnet-channel distance. Additional numerical studies of the geometric effects of the U-turn reveal that increasing the outlet-branch width of the U-turn can significantly enhance the diamagnetic particle separation in ferrofluids. - Highlights: • Particles are focused and separated in the two branches of a U-shaped microchannel. • Negative magnetophoretic motion in ferrofluids causes the particle deflection. • A 3D numerical model is developed to simulate the particle separation. • Multiple parametric effects are studied both experimentally and numerically. • Increasing the outlet-branch width significantly enhances the particle separation.

An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

DEFF Research Database (Denmark)

Hansen, Soren; Schmidt, Vivi; Steffensen, Maria Abildgaard

2008-01-01

characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which......Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other...... innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were...

The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan.

Science.gov (United States)

Mizanbayeva, Sulushash; Smits, Henk L; Zhalilova, Katya; Abdoel, Theresia H; Kozakov, Stanislaw; Ospanov, Kenes S; Elzer, Philip H; Douglas, James T

2009-09-01

Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers > or = 1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high sensitivity of these assays. The strong reactivity in the LFA observed for the majority (92.1%) of the samples from the patients with culture-confirmed brucellosis together with the user-friendliness of the assay procedure makes the LFA ideal for the confirmation of brucellosis in endemic areas in Kazakhstan. The Rose Bengal test lacked sensitivity in particular for patients with chronic brucellosis therefore limiting its value as a quick screening assay. The study emphasizes the importance of the LFA as a useful, rapid, and easy-to-perform tool in the diagnostic testing of brucellosis.

USE OF AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA TO DETECT ANTIBODIES IN AYU (Plecogiossus altivelis VACCINATED BY IMMERSION ADMINISTRATION

Directory of Open Access Journals (Sweden)

. Sukenda

2007-05-01

Full Text Available ABSTRACTAn indirect enzyme-linked immunosorbent assay (ELISA was used to detect serum antibody in ayu, Plecoglossus altivelis, immunized against Pseudomonasplecoglossicida by immersion vaccination.  First, the procedure of the ELISA was optimized and the sensitivity was checked.  Secondly, the formalin-killed cells (FKC of P. plecoglossicida was administered to ayu by immersion vaccination.  Two weeks after vaccination, fish were divided into two groups, one group was given booster.  The level of specific antibody production of both boostered and vaccinated only fish were statistically higher than unvaccinated control fish at the time of each blood collection.  However, the differences between the boostered and vaccinated only fish were not statistically significant.Keywords :  immunization, Pseudomonas plecoglossicida, ayu, ELISA ABSTRAKIndirect enzyme-linked immunosorbent assay (ELISA digunakan untuk mendeteksi antibodi pada ayu, Plecoglossus altivelis, yang diimunisasi dengan cara perendaman untuk melawan infeksi Pseudomonas plecoglossicida.  Pertama, prosedur ELISA dioptimasikan dan sensitivitas dari metode ini juga diperiksa.  Kemudian, bakteri Plecoglossus altivelis yang sudah dimatikan dengan formalin diberikan ke ikan ayu dengan vaksinasi perendaman.  Dua minggu setelah vaksinasi, ikan dibagi menjadi dua kelompok, satu kelompok diberi vaksinasi kedua.  Produksi antibodi spesifik dari ikan-ikan yang divaksinasi satu kali dengan vaksinasi dua kaii secara statistik lebih tinggi dibandingkan dengan control.  Akan tetapi, tidak ada perbedaan produksi antibodi antara ikan yarig divaksanisi satu kali dengan divaksinasi dua kali.Kata kunci :  imunisasi, Pseudomonasplecoglossicida, ayu, ELISA

Hyaluronic Acid Assays

DEFF Research Database (Denmark)

Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

2015-01-01

BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

In vivo continuous and simultaneous monitoring of brain energy substrates with a multiplex amperometric enzyme-based biosensor device

NARCIS (Netherlands)

De Lima Braga Lopes Cordeiro, Carlos; de Vries, M.G.; Ngabi, W; Oomen, P.E.; Cremers, T.I.F.H.; Westerink, B.H.C.

2015-01-01

Enzyme-based amperometric biosensors are widely used for monitoring key biomarkers. In experimental neuroscience there is a growing interest in in vivo continuous and simultaneous monitoring of metabolism-related biomarkers, like glucose, lactate and pyruvate. The use of multiplex biosensors will

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid.

Science.gov (United States)

Aw, Alex; Fritz, Marshall; Napoline, Jonathan W; Pollet, Pamela; Liotta, Charles L

2017-11-15

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the "scaling out" strategy as opposed to the traditional "scaling up". Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with "mixing" and "linear" microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology's competitiveness, sustainability, and versatility in their research.

Effects of different enzyme treatments in extraction of total folate from infant formula, baby foods and other food products prior to microbiological assay and radioassay

International Nuclear Information System (INIS)

De Souza, S.C.

1988-01-01

Four different enzyme treatments-conjugase alone, conjugase and alpha-amylase, conjugase and Pronase reg-sign and a triple enzyme combination of conjugase, Pronase reg-sign and alpha-amylase were applied in the extraction of total folate from infant formula, baby foods and various other foods by microbiological and radioassay methods. Significant increases (P < 0.05) in measurable folate were obtained using the triple enzyme system in spinach, Camembert cheese, soy-based infant formula and cereal-based, meat-based and fruit-based infant foods over the use of conjugase alone by the microbiological method. Increases were also observed in many of the same foods using Pronase reg-sign or alpha-amylase in addition to conjugase alone. Increases obtained by microbiological assay were confirmed by radioassay in a number of foods studied

Recycle bioreactor for bioethanol production from wheat starch. 1. Cold enzyme hydrolysis

Energy Technology Data Exchange (ETDEWEB)

Lang, X.; Hill, G.A.; MacDonald, D.G. [Department of Chemical Engineering, Saskatchewan (Canada)

2001-06-01

A 5 L membrane bioreactor system has been designed and operated at low temperature to hydrolyze starch granules directly to sugars using barley {alpha}-amylase. The system includes a temperature and pH controlled, well-mixed bioreactor; microfilters to separate and recycle granules; and ultrafilters to separate and recycle enzyme molecules. Operation in batch mode demonstrated similar kinetics and low productivity observed earlier in shake flasks, whereas continuous flow operation was not successful due to enzyme inhibition and degradation. Sequential batch mode operation, involving filtration after each batch hydrolysis, produced optimum productivity measured at 0.16 grams of starch granules hydrolyzed per gram of enzyme per hour for more than 100 hours of operation. (author)

Epidural blood flow and regression of sensory analgesia during continuous postoperative epidural infusion of bupivacaine

DEFF Research Database (Denmark)

Mogensen, T; Højgaard, L; Scott, N B

1988-01-01

Epidural blood flow was measured in seven patients undergoing elective abdominal surgery during combined lumbar epidural and general anesthesia. After an initial dose of 20 ml plain bupivacaine 0.5%, a continuous epidural infusion of bupivacaine 0.5% (8 ml/hr) was given for 16 hours for postopera......Epidural blood flow was measured in seven patients undergoing elective abdominal surgery during combined lumbar epidural and general anesthesia. After an initial dose of 20 ml plain bupivacaine 0.5%, a continuous epidural infusion of bupivacaine 0.5% (8 ml/hr) was given for 16 hours...... surgery, and 8, 12, and 16 hours later during the continuous infusion. Initial blood flow was 6.0 +/- 0.7 ml/min per 100 g tissue (mean +/- SEM). After epidural bupivacaine, blood flow increased in all seven patients to 7.4 +/- 0.7 ml (P less than 0.02). Initial level of sensory analgesia was T4.5 +/- 0...... than 0.03) in the other five patients as the level of sensory analgesia regressed postoperatively. These data suggest that changes in epidural blood flow during continuous epidural infusion of bupivacaine, and thus changes in rates of vascular absorption of bupivacaine from the epidural space, may...

Serum biotin in Japanese children: Enzyme-linked immunosorbent assay measurement.

Science.gov (United States)

Wakabayashi, Kenji; Kodama, Hiroko; Ogawa, Eishin; Sato, Yasuhiro; Motoyama, Kahoko; Suzuki, Mitsuyoshi

2016-09-01

Biotin deficiency has been reported in Japanese infants fed special formulas for medical reasons, including those with milk allergy and congenital metabolic diseases, because these formulas contain little biotin. Serum biotin measurement is useful for diagnosing biotin deficiency. We applied a simple and rapid method to analyze serum biotin, and established normal ranges for children and adults. Serum biotin in 188 healthy Japanese children aged 0-4 years and in 25 healthy adults was analyzed using a Biotin ELISA Kit (immundiagnostik). The effects of various conditions on the measurement of serum biotin were also examined. Median biotin in children aged 0-4 years was 10.4 ng/dL (IQR, 7.9-13.4 ng/dL), and that in adults was 12.9 ng/dL (IQR, 10.8-15.8 ng/dL). Normal range was 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults (calculated using two-sided 95%CI). Measurements obtained with this method were not affected by frozen storage, freeze-thaw, or hemolysis, indicating that serum biotin can be analyzed accurately under these conditions, with a possible application to plasma samples. Serum biotin was significantly lower in children than in adults, with the normal range being 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults. This simple and accurate enzyme-linked immunosorbent assay method is useful for diagnosing biotin deficiency. © 2016 The Authors. Pediatrics International published by John Wiley & Sons Australia, Ltd on behalf of Japan Pediatric Society.

Continuous Flow Nucleophilic Aromatic Substitution with Dimethylamine Generated in Situ by Decomposition of DMF

DEFF Research Database (Denmark)

Petersen, Trine P; Larsen, Anders Foller; Ritzén, Andreas

2013-01-01

A safe, practical, and scalable continuous flow protocol for the in situ generation of dimethylamine from DMF followed by nucleophilic aromatic substitution of a broad range of aromatic and heteroaromatic halides is reported.......A safe, practical, and scalable continuous flow protocol for the in situ generation of dimethylamine from DMF followed by nucleophilic aromatic substitution of a broad range of aromatic and heteroaromatic halides is reported....

Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

Science.gov (United States)

Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

2005-09-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

Science.gov (United States)

Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

2005-01-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

Detection of Plasmodium Aldolase Using a Smartphone and Microfluidic Enzyme Linked Immunosorbent Assay

Directory of Open Access Journals (Sweden)

Nikhil S. Gopal

2017-01-01

Full Text Available Background. Malaria control efforts are limited in rural areas. A low-cost system to monitor response without the use of electricity is needed. Plasmodium aldolase is a malaria biomarker measured using enzyme linked immunosorbent assay (ELISA techniques. A three-part system using ELISA was developed consisting of a microfluidic chip, hand crank centrifuge, and a smartphone. Methods. A circular microfluidic chip was fabricated using clear acrylic and a CO2 laser. A series of passive valves released reagents at precise times based upon centrifugal force. Color change was measured via smartphone camera using an application programmed in Java. The microchip was compared to a standard 96-well sandwich ELISA. Results. Results from standard ELISA were compared to microchip at varying concentrations (1–10 ng/mL. Over 15 different microfluidic patterns were tested, and a final prototype of the chip was created. The prototype microchip was compared to standard sandwich ELISA (n=20 using samples of recombinant aldolase. Color readings of standard ELISA and microfluidic microchip showed similar results. Conclusion. A low-cost microfluidic system could detect and follow therapeutic outcomes in rural areas and identify resistant strains.

Nanoparticle-based assays in automated flow systems: A review

Energy Technology Data Exchange (ETDEWEB)

Passos, Marieta L.C. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Pinto, Paula C.A.G., E-mail: ppinto@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Santos, João L.M., E-mail: joaolms@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Saraiva, M. Lúcia M.F.S., E-mail: lsaraiva@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Araujo, André R.T.S. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda, Av. Dr. Francisco de Sá Carneiro, n° 50, 6300-559 Guarda (Portugal)

2015-08-19

Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section.

Nanoparticle-based assays in automated flow systems: A review

International Nuclear Information System (INIS)

Passos, Marieta L.C.; Pinto, Paula C.A.G.; Santos, João L.M.; Saraiva, M. Lúcia M.F.S.; Araujo, André R.T.S.

2015-01-01

Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section

Enzyme-Linked Immunosorbent Assay To Differentiate the Antibody Responses of Animals Infected with Brucella Species from Those of Animals Infected with Yersinia enterocolitica O9

OpenAIRE

Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

2003-01-01

Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

Science.gov (United States)

Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

2015-04-21

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

Science.gov (United States)

Helmerhorst, Eva J; Wei, Guoxian

2014-05-05

Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein e(rns) for serologic diagnosis of pestivirus infections in swine

NARCIS (Netherlands)

Langedijk, J.P.; Middel, W.G.; Meloen, R.H.; Kramps, J.A.; Smit, de J.A.

2001-01-01

Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure

[Advances on enzymes and enzyme inhibitors research based on microfluidic devices].

Science.gov (United States)

Hou, Feng-Hua; Ye, Jian-Qing; Chen, Zuan-Guang; Cheng, Zhi-Yi

2010-06-01

With the continuous development in microfluidic fabrication technology, microfluidic analysis has evolved from a concept to one of research frontiers in last twenty years. The research of enzymes and enzyme inhibitors based on microfluidic devices has also made great progress. Microfluidic technology improved greatly the analytical performance of the research of enzymes and enzyme inhibitors by reducing the consumption of reagents, decreasing the analysis time, and developing automation. This review focuses on the development and classification of enzymes and enzyme inhibitors research based on microfluidic devices.

Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

Science.gov (United States)

Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

2010-10-01

There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

Evaluation of electrochemiluminescene immunoassay and enzyme-linked immunosorbent assay for serum HBsAb detection

International Nuclear Information System (INIS)

Ma Caiyun; Jiang Li; Ge Gaoxia; Zhang Xiaojie

2005-01-01

Electrochemiluminescene immunoassay (ECLIA) and enzyme-linked immunosorbent (ELISA) were used to detect the different concentrations of serum HBsAb, in order to compare the results of ECLIA and ELISA. The result showed that intra-assay coefficient of variation of ECLIA was about 0.95% for high value, 1.13% for mean values and 2.63% for low value, while that of ELISA was about 5.76%, 12.8% and 15.9%, respectively. The interassay coefficient of ECLIA was about 4.03% for high values, 4.93% for mean values and 7.34% for low values, while that of ELISA was about 10.1%, 19.6% and 25.2%, respectively. The analytical sensitivity of ECLIA was about 4.0IU/L, while that of ELISA is about 19.0IU/L. Only in 3 samples, the results measured by ECLIA and ELISA were different (ECLIA: positive; ELISA: negative) among 165 samples. It is concluded that the met hod of ECLIA is more efficient than ELISA for detection of HBsAb in serum. (authors)

Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

International Nuclear Information System (INIS)

Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

1981-01-01

Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10 5 and greater than or equal to 3 X 10 6 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10 5 and greater than or equal to10 4 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

State of the art of aerobic granulation in continuous flow bioreactors.

Science.gov (United States)

Kent, Timothy R; Bott, Charles B; Wang, Zhi-Wu

In the wake of the success of aerobic granulation in sequential batch reactors (SBRs) for treating wastewater, attention is beginning to turn to continuous flow applications. This is a necessary step given the advantages of continuous flow treatment processes and the fact that the majority of full-scale wastewater treatment plants across the world are operated with aeration tanks and clarifiers in a continuous flow mode. As in SBRs, applying a selection pressure, based on differences in either settling velocity or the size of the biomass, is essential for successful granulation in continuous flow reactors (CFRs). CFRs employed for aerobic granulation come in multiple configurations, each with their own means of achieving such a selection pressure. Other factors, such as bioaugmentation and hydraulic shear force, also contribute to aerobic granulation to some extent. Besides the formation of aerobic granules, long-term stability of aerobic granules is also a critical issue to be addressed. Inorganic precipitation, special inocula, and various operational optimization strategies have been used to improve granule long-term structural integrity. Accumulated studies reviewed in this work demonstrate that aerobic granulation in CFRs is capable of removing a wide spectrum of contaminants and achieving properties generally comparable to those in SBRs. Despite the notable research progress made toward successful aerobic granulation in lab-scale CFRs, to the best of our knowledge, there are only three full-scale tests of the technique, two being seeded with anammox-supported aerobic granules and the other with conventional aerobic granules; two other process alternatives are currently in development. Application of settling- or size-based selection pressures and feast/famine conditions are especially difficult to implement to these and similar mainstream systems. Future research efforts needs to be focused on the optimization of the granule-to-floc ratio, enhancement of

The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan

NARCIS (Netherlands)

Mizanbayeva, Sulushash; Smits, Henk L.; Zhalilova, Katya; Abdoel, Theresia H.; Kozakov, Stanislaw; Ospanov, Kenes S.; Elzer, Philip H.; Douglas, James T.

2009-01-01

Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers > or = 1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high

A New Enzyme-linked Sorbent Assay (ELSA) to Quantify Syncytiotrophoblast Extracellular Vesicles in Biological Fluids.

Science.gov (United States)

Göhner, Claudia; Weber, Maja; Tannetta, Dionne S; Groten, Tanja; Plösch, Torsten; Faas, Marijke M; Scherjon, Sicco A; Schleußner, Ekkehard; Markert, Udo R; Fitzgerald, Justine S

2015-06-01

The pregnancy-associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are available and accepted, though imperfect. For this purpose, we aimed to provide an enzyme-linked sorbent assay (ELSA) to quantify STBEV in fluid samples based on their microvesicle characteristics and placental origin. Ex vivo placenta perfusion provided standards and samples for the STBEV quantification. STBEV were captured by binding of extracellular phosphatidylserine to immobilized annexin V. The membranous human placental alkaline phosphatase on the STBEV surface catalyzed a colorimetric detection reaction. The described ELSA is a rapid and simple method to quantify STBEV in diverse liquid samples, such as blood or perfusion suspension. The reliability of the ELSA was proven by comparison with nanoparticle tracking analysis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

Energy Technology Data Exchange (ETDEWEB)

Booth, J C; Hannington, G; Bakir, T M.F.; Stern, H; Kangro, H; Griffiths, P D; Heath, R B [Saint George' s Hospital Medical School, London (UK); Saint Bartholomew' s Hospital, London (UK))

1982-12-01

The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems.

An electric detection of immunoglobulin G in the enzyme-linked immunosorbent assay using an indium oxide nanoparticle ion-sensitive field-effect transistor

International Nuclear Information System (INIS)

Lee, Dongjin; Cui, Tianhong

2012-01-01

Semiconducting nanoparticle ion-sensitive field-effect transistors (ISFETs) are used to detect immunoglobulin G (IgG) in the conventional enzyme-linked immunosorbent assay (ELISA). Indium oxide and silica nanoparticles were layer-by-layer self-assembled with the oppositely charged polyelectrolyte as the electrochemical transducer and antibody immobilization site, respectively. The assay was conducted on a novel platform of indium oxide nanoparticle ISFETs, where the electric signals are generated in response to the concentration of target IgG using the labeled detecting antibody. The sandwiched ELISA structure catalyzed the conversion of the acidic substrate into neutral substance with the aid of horseradish peroxidase. The pH change in the substrate solution was detected by nanoparticle ISFETs. Normal rabbit IgG was used as a model antigen whose detection limit of 0.04 ng ml −1 was found. The facile electric detection in the conventional assay through the semiconducting nanoparticle ISFET has potential applications as a point-of-care detection or a sensing element in a lab-on-a-chip system

Antibody-based enzyme-linked lectin assay (ABELLA) for the sialylated recombinant human erythropoietin present in culture supernatant.

Science.gov (United States)

Kim, Hyoung Jin; Lee, Seung Jae; Kim, Hong-Jin

2008-11-04

The terminal sialic acid of human erythropoietin (hEPO) is essential for in vivo activity. The current resorcinol and HPLC methods for analyzing alpha2,3-linked sialic acid require more than a microgram of purified rhEPO, and purification takes a great deal of time and labor. In this study, we assessed the use of an antibody-based enzyme-linked lectin assay (ABELLA) for analyzing non-purified recombinant hEPO (rhEPO). The major problem of this method was the high background due to terminal sialylation of components of the assay (antibody and bovine serum albumin) other than rhEPO. To solve this problem, we used a monoclonal antibody (Mab 287) to capture the rhEPO, and oxidized the bovine serum albumin used for blocking with meta-periodate. The sialic acid content of non-purified rhEPO measured by ABELLA was similar to that obtained by the resorcinol method on purified rhEPO. ABELLA has advantages such as adaptability and need for minimal amounts of rhEPO (40 ng/ml). Our observations suggest that ABELLA should reduce the time and labor needed to improve culture conditions so as to increase protein sialylation, and also facilitate the study of sialylation mechanisms.

Hydrothermal Processing of Macroalgal Feedstocks in Continuous-Flow Reactors

Energy Technology Data Exchange (ETDEWEB)

Elliott, Douglas C.; Hart, Todd R.; Neuenschwander, Gary G.; Rotness, Leslie J.; Roesijadi, Guri; Zacher, Alan H.; Magnuson, Jon K.

2014-02-03

Wet macroalgal slurries have been converted into a biocrude by hydrothermal liquefaction (HTL) in a bench-scale continuous-flow reactor system. Carbon conversion to a gravity-separable oil product of 58.8% was accomplished at relatively low temperature (350 °C) in a pressurized (subcritical liquid water) environment (20 MPa) when using feedstock slurries with a 21.7% concentration of dry solids. As opposed to earlier work in batch reactors reported by others, direct oil recovery was achieved without the use of a solvent, and biomass trace mineral components were removed by processing steps so that they did not cause processing difficulties. In addition, catalytic hydrothermal gasification (CHG) was effectively applied for HTL byproduct water cleanup and fuel gas production from water-soluble organics. Conversion of 99.2% of the carbon left in the aqueous phase was demonstrated. Finally, as a result, high conversion of macroalgae to liquid and gas fuel products was found with low levels of residual organic contamination in byproduct water. Both process steps were accomplished in continuous-flow reactor systems such that design data for process scale-up was generated.

Enzymic hydrolysis of starch in continuous alcoholic fermentation

Energy Technology Data Exchange (ETDEWEB)

Yarovenko, V.L.; Pykhova, S.V.; Ustinnikov, B.A.; Lazareva, A.N.; Makeev, D.M.

1965-01-01

Fermentations were conducted on a plant scale, using starch of various origins, e.g., potatoes, wheat, and other cereals, and as enzyme source a number of strains of Aspergillus oryzae, A. awamori, A. usamii, A. niger, A. batatae, and Bacillus mesentericus. The starches were broken down to a molecular weight between 900 and 1600; time requirements differed from those commonly known. Comparison of these enzymic actions to that of standard malt discloses that in breaking down a potato starch from a molecular weight of 268,000 to one of 1353 to 1556, the malt and A. Oryzae require 1 hour, whereas A. awamori and B. mesentericus require 18, and a different strain of A. awamori requires 24 hours.

Biosensors for the determination of environmental inhibitors of enzymes

Energy Technology Data Exchange (ETDEWEB)

Evtugyn, Gennadii A; Budnikov, Herman C [Kazan State University, Kazan (Russian Federation); Nikolskaya, Elena B [I.M. Sechenov Institute of Evolution Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg (Russian Federation)

1999-12-31

Characteristic features of functioning and practical application of enzyme-based biosensors for the determination of environmental pollutants as enzyme inhibitors are considered with special emphasis on the influence of the methods used for the measurement of the rates of enzymic reactions, of enzyme immobilisation procedure and of the composition of the reaction medium on the analytical characteristics of inhibitor assays. The published data on the development of biosensors for detecting pesticides and heavy metals are surveyed. Special attention is given to the use of cholinesterase-based biosensors in environmental and analytical monitoring. The approaches to the estimation of kinetic parameters of inhibition are reviewed and the factors determining the selectivity and sensitivity of inhibitor assays in environmental objects are analysed. The bibliography includes 195 references.

Biosensors for the determination of environmental inhibitors of enzymes

International Nuclear Information System (INIS)

Evtugyn, Gennadii A; Budnikov, Herman C; Nikolskaya, Elena B

1999-01-01

Characteristic features of functioning and practical application of enzyme-based biosensors for the determination of environmental pollutants as enzyme inhibitors are considered with special emphasis on the influence of the methods used for the measurement of the rates of enzymic reactions, of enzyme immobilisation procedure and of the composition of the reaction medium on the analytical characteristics of inhibitor assays. The published data on the development of biosensors for detecting pesticides and heavy metals are surveyed. Special attention is given to the use of cholinesterase-based biosensors in environmental and analytical monitoring. The approaches to the estimation of kinetic parameters of inhibition are reviewed and the factors determining the selectivity and sensitivity of inhibitor assays in environmental objects are analysed. The bibliography includes 195 references.

A multiwell format assay for heparanase.

Science.gov (United States)

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

Graphene-based battery electrodes having continuous flow paths

Science.gov (United States)

Zhang, Jiguang; Xiao, Jie; Liu, Jun; Xu, Wu; Li, Xiaolin; Wang, Deyu

2014-05-24

Some batteries can exhibit greatly improved performance by utilizing electrodes having randomly arranged graphene nanosheets forming a network of channels defining continuous flow paths through the electrode. The network of channels can provide a diffusion pathway for the liquid electrolyte and/or for reactant gases. Metal-air batteries can benefit from such electrodes. In particular Li-air batteries show extremely high capacities, wherein the network of channels allow oxygen to diffuse through the electrode and mesopores in the electrode can store discharge products.

Immunocapture loop-mediated isothermal amplification assays for the detection of canine parvovirus.

Science.gov (United States)

Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

2017-11-01

A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10 -1 , 10 -1 , and 10 -1 TCID 50 /mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV. Copyright © 2017 Elsevier B.V. All rights reserved.

Competitive-IgY- Enzyme Linked Immuno Sorbent Assay (CIgY-ELISA to detect the cytokinins in Gerbera jamesonii plantlets

Directory of Open Access Journals (Sweden)

Cleiton Mateus Sousa

2011-08-01

Full Text Available A competitive hyper-immune yolk Immunoglobulin Y - Enzyme Linked Immune Sorbent Assay (CIgY-ELISA, was developed as an alternative method to detect zeatin and 2ip in plantlets of gerbera. The endogenous level of hormones in the plantlets in vitro of gerbera with one or six weeks after replication was determined with competitive IgY-ELISA set to detect between 1 and 100 pmoles of plant hormone for each 1.0 g tissue. The plantlets of six weeks presented sprouts and root, while the plantlets of one week presented only sprouts. The CIgY-ELISA was set with high independent variables values of sensitivity/specificity of 96/89 % for zeatin and 94/78 % for 2ip, with high values of reproducibility (up to 90 % for both the cytokinins. Zeatin content varied from 2.2 to 2.8 pmoles.g-1 and from 2.7 to 3.3 pmoles.g-1 on the plantlet with one and six weeks, respectively. The 2ip content did not vary and was detected near the detection limit in all the assays. It was concluded that the observed capabilities of CIgY-ELISA were putative and the competitive assay was a highly robust and stable method, which could be used for the studies on plant physiology for endogenous cytokinins.

Development of an enzyme-linked immunosorbent assay for residue analysis of the insecticide emamectin benzoate in agricultural products.

Science.gov (United States)

Kondo, Mika; Yamashita, Hiroshi; Uchigashima, Mikiko; Kono, Takeshi; Takemoto, Toshihide; Fujita, Masahiro; Saka, Machiko; Iwasa, Seiji; Ito, Shigekazu; Miyake, Shiro

2009-01-28

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for the analysis of emamectin residues in agricultural products was developed using a prepared mouse monoclonal antibody. The working range was 0.3-3.0 ng/mL, and the 50% inhibition concentration (IC(50)) was 1.0 ng/mL. The assay was sufficiently sensitive for analysis of the maximum residue limits in agricultural products in Japan (>0.1 microg/g). Emamectin residues contain the following metabolites: the 4''-epi-amino analogue, the 4''-epi-(N-formyl)amino analogue, the 4''-epi-(N-formyl-N-methyl)amino analogue, and the 8,9-Z isomer. The dc-ELISA reacted with these compounds at ratios of 113, 55, 38, and 9.1% of the IC(50) value of emamectin benzoate. Seven kinds of vegetables were spiked with emamectin benzoate at concentrations of 15-300 ng/g, and the recoveries were 91-117% in the dc-ELISA. The dc-ELISA results agreed reasonably well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using spiked samples and actual (incurred) samples. The results indicate that the dc-ELISA was useful for the analysis of emamectin benzoate residues in agricultural products.

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus.

Science.gov (United States)

Kardi, V; Szegletes, E; Perényi, T; Pergel, I; Smal, Z

1990-01-01

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.

Aldolase catalyzed L-phenylserine synthesis in a slug-flow microfluidic system - Performance and diastereoselectivity studies

NARCIS (Netherlands)

Čech, J.; Hessel, V.; Přibyl, M.

2017-01-01

We study synthesis of . L-phenylserine catalyzed by the enzyme . L-threonine aldolase in a slug-flow microfluidic system. Slug-flow arrangement allows for the continuous refilling of sparingly soluble substrate (benzaldehyde) into an aqueous reaction mixture. We identified suitable composition of an

Rotor assembly and assay method

Science.gov (United States)

Burtis, C.A.; Johnson, W.F.; Walker, W.A.

1993-09-07

A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

Diazo compounds in continuous-flow technology.

Science.gov (United States)

Müller, Simon T R; Wirth, Thomas

2015-01-01

Diazo compounds are very versatile reagents in organic chemistry and meet the challenge of selective assembly of structurally complex molecules. Their leaving group is dinitrogen; therefore, they are very clean and atom-efficient reagents. However, diazo compounds are potentially explosive and extremely difficult to handle on an industrial scale. In this review, it is discussed how continuous flow technology can help to make these powerful reagents accessible on large scale. Microstructured devices can improve heat transfer greatly and help with the handling of dangerous reagents safely. The in situ formation and subsequent consumption of diazo compounds are discussed along with advances in handling diazomethane and ethyl diazoacetate. The potential large-scale applications of a given methodology is emphasized. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nuclear assay of coal. Volume 6. Mass flow devices for coal handling

International Nuclear Information System (INIS)

Anon.

1979-01-01

The mass of coal entering the boiler per unit time is an essential parameter for determinig the total rate of heat input. The mass flow rate of coal on a conveyor belt is generally determined as a product of the instantaneous mass of material on a short section of the belt and the belt velocity. Belt loading could be measured by conventional transducers incorporating mechanical or electromechanical weighers or by gamma-ray attenuation gauge. This report reviews the state of the art in mass flow devices for coal handling. The various methods are compared and commented upon. Special design issues are discussed relative to incorporating a mass flow measuring device in a Continuous On-Line Nuclear Analysis of Coal (CONAC) system

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

Science.gov (United States)

Garver, W S; Kemp, J D; Kuehn, G D

1992-12-01

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

"Batch" kinetics in flow: online IR analysis and continuous control.

Science.gov (United States)

Moore, Jason S; Jensen, Klavs F

2014-01-07

Currently, kinetic data is either collected under steady-state conditions in flow or by generating time-series data in batch. Batch experiments are generally considered to be more suitable for the generation of kinetic data because of the ability to collect data from many time points in a single experiment. Now, a method that rapidly generates time-series reaction data from flow reactors by continuously manipulating the flow rate and reaction temperature has been developed. This approach makes use of inline IR analysis and an automated microreactor system, which allowed for rapid and tight control of the operating conditions. The conversion/residence time profiles at several temperatures were used to fit parameters to a kinetic model. This method requires significantly less time and a smaller amount of starting material compared to one-at-a-time flow experiments, and thus allows for the rapid generation of kinetic data. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of enzymes on anaerobic digestion of primary sludge and septic tank performance.

Science.gov (United States)

Diak, James; Örmeci, Banu; Kennedy, Kevin J

2012-11-01

Enzyme additives are believed to improve septic tank performance by increasing the hydrolysis and digestion rates and maintaining a healthy microbial population. Previous studies reported mixed results on the effectiveness of enzymes on mesophilic and thermophilic digestion, and it is not clear whether enzymes would be effective under septic tank conditions where there is no heating or mixing, quantities of enzymes added are small, and they can be washed out quickly. In this study, batch reactors and continuous-flow reactors designed and operated as septic tanks were used to evaluate whether enzymatic treatment would increase the hydrolysis and digestion rates in primary sludge. Total solids, volatile solids, total suspended solids, total and soluble chemical oxygen demand, concentrations of protein, carbohydrate, ammonia and volatile acids in sludge and effluent samples were measured to determine the differences in digestion rates in the presence and absence of enzymes. Overall, no significant improvement was observed in enzyme-treated reactors compared with the control reactors.

Assay for mutagenesis in heterozygous diploid human lymphoblasts

Science.gov (United States)

Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

1981-01-01

An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

Science.gov (United States)

Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

2014-03-26

A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integrating continuous stocks and flows into state-and-transition simulation models of landscape change

Science.gov (United States)

Daniel, Colin J.; Sleeter, Benjamin M.; Frid, Leonardo; Fortin, Marie-Josée

2018-01-01

State-and-transition simulation models (STSMs) provide a general framework for forecasting landscape dynamics, including projections of both vegetation and land-use/land-cover (LULC) change. The STSM method divides a landscape into spatially-referenced cells and then simulates the state of each cell forward in time, as a discrete-time stochastic process using a Monte Carlo approach, in response to any number of possible transitions. A current limitation of the STSM method, however, is that all of the state variables must be discrete.Here we present a new approach for extending a STSM, in order to account for continuous state variables, called a state-and-transition simulation model with stocks and flows (STSM-SF). The STSM-SF method allows for any number of continuous stocks to be defined for every spatial cell in the STSM, along with a suite of continuous flows specifying the rates at which stock levels change over time. The change in the level of each stock is then simulated forward in time, for each spatial cell, as a discrete-time stochastic process. The method differs from the traditional systems dynamics approach to stock-flow modelling in that the stocks and flows can be spatially-explicit, and the flows can be expressed as a function of the STSM states and transitions.We demonstrate the STSM-SF method by integrating a spatially-explicit carbon (C) budget model with a STSM of LULC change for the state of Hawai'i, USA. In this example, continuous stocks are pools of terrestrial C, while the flows are the possible fluxes of C between these pools. Importantly, several of these C fluxes are triggered by corresponding LULC transitions in the STSM. Model outputs include changes in the spatial and temporal distribution of C pools and fluxes across the landscape in response to projected future changes in LULC over the next 50 years.The new STSM-SF method allows both discrete and continuous state variables to be integrated into a STSM, including interactions between

Direct Human Papillomavirus E6 Whole-Cell Enzyme-Linked Immunosorbent Assay for Objective Measurement of E6 Oncoproteins in Cytology Samples

OpenAIRE

Yang, Yi-Shan; Smith-McCune, Karen; Darragh, Teresa M.; Lai, Yvonne; Lin, Ju-Hwa; Chang, Ting-Chang; Guo, Hsiao-Yun; Kesler, Tiea; Carter, Alicia; Castle, Philip E.; Cheng, Shuling

2012-01-01

A novel, whole-cell enzyme-linked immunosorbent assay (ELISA) based on a non-type-specific anti-human papillomavirus (HPV) E6 antibody was tested on 182 residual cytological specimens. For samples with a designation of more severe than cervical intraepithelial neoplasia grade 3 (CIN3+), 83% tested positive for E6; in a subset with paired testing for E6 ELISA and HPV DNA, 72% tested E6 positive and 92% tested high-risk (HR)-HPV DNA positive (P = 0.2). Among the women with a less than CIN3 diag...

Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program

DEFF Research Database (Denmark)

Chung, Wen Bin; Sørensen, Karl Johan; Liao, Pei Chih

2002-01-01

Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...... in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased....

Application of a microcystin extraction method specific for enzyme inhibition assays

International Nuclear Information System (INIS)

Sevilla Miguel, E.; Simienk, H.; Calvin Tienza, V.; Razquin Casquero, P.; Peleato Sanchez, M. L.; Mata Vallespin, L.

2009-01-01

A method for the determination of intracellular and dissolved microcystins in non treated water is proposed. The results obtained with this method, based on a phosphatase inhibition assay, are compared with those for HPLC- UV. Potential interferences of the phosphatase inhibition assays like pigments or the endogenous phosphatase activity present in cyanobacteria did not have any adverse effect on assay results. Besides, the recovery of microcystins in field samples with the proposed method was found to be high than 90% in all tested samples. A number of samples from different origins and appearances were also analyzed for their microcystin content. (Author) 27 refs

Lignin-degrading enzyme activities.

Science.gov (United States)

Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

2012-01-01

Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

DEFF Research Database (Denmark)

Leeming, Diana Julie; He, Y.; Veidal, S. S.

2011-01-01

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) an...